Modulation of angiopoietin-like 3 expression

ABSTRACT

Provided herein are methods, compounds, and compositions for reducing expression of an ANGPTL3 mRNA and protein in an animal. Also provided herein are methods, compounds, and compositions for reducing plasma lipids, plasma glucose and atherosclerotic plaques in an animal. Such methods, compounds, and compositions are useful to treat, prevent, delay, or ameliorate any one or more of cardiovascular disease or metabolic disease, or a symptom thereof.

CROSS REFERENCED TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/149,715 filed Jan. 7, 2014 which is a divisional of U.S. applicationSer. No. 13/520,997 filed Oct. 4, 2012, issued as U.S. Pat. No.8,653,047 which is a U.S. National Phase filing under 35 U.S.C. 371claiming priority to International Serial No. PCT/US2011/020606 filedJan. 7, 2011, which is the non-provisional application of U.S.Provisional Application No. 61/293,604, filed Jan. 8, 2010, each ofwhich is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0120USC1SEQ.txt, created on Aug. 7, 2015 which is 76 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are methods, compounds, and compositions for reducingexpression of angiopoietin-like 3 (ANGPTL3) mRNA and protein in ananimal. Also, provided herein are methods, compounds, and compositionshaving an ANGPTL3 inhibitor for reducing ANGPTL3 related diseases orconditions in an animal. Such methods, compounds, and compositions areuseful, for example, to treat, prevent, delay or ameliorate any one ormore of cardiovascular disease or metabolic syndrome, or a symptomthereof, in an animal.

BACKGROUND

Diabetes and obesity (sometimes collectively referred to as “diabesity”)are interrelated in that obesity is known to exacerbate the pathology ofdiabetes and greater than 60% of diabetics are obese. Most human obesityis associated with insulin resistance and leptin resistance. In fact, ithas been suggested that obesity may have an even greater impact oninsulin action than diabetes itself (Sindelka et al., Physiol Res.,2002, 51, 85-91). Additionally, several compounds on the market for thetreatment of diabetes are known to induce weight gain, a veryundesirable side effect to the treatment of this disease.

Cardiovascular disease is also interrelated to obesity and diabetes.Cardiovascular disease encompasses a wide variety of etiologies and hasan equally wide variety of causative agents and interrelated players.Many causative agents contribute to symptoms such as elevated plasmalevels of cholesterol, including non-HDL cholesterol, as well as otherlipid-related disorders. Such lipid-related disorders, generallyreferred to as dyslipidemia, include hyperlipidemia,hypercholesterolemia and hypertriglyceridemia among other indications.Elevated non-HDL cholesterol is associated with atherogenesis and itssequelae, including cardiovascular diseases such as arteriosclerosis,coronary artery disease, myocardial infarction, ischemic stroke, andother forms of heart disease. These rank as the most prevalent types ofillnesses in industrialized countries. Indeed, an estimated 12 millionpeople in the United States suffer with coronary artery disease andabout 36 million require treatment for elevated cholesterol levels.

Epidemiological and experimental evidence has shown that high levels ofcirculating triglyceride (TG) can contribute to cardiovascular diseaseand a myriad of metabolic disorders (Valdivielso et al., 2009,Atherosclerosis. 207(2):573-8; Zhang et al., 2008, Circ Res. 1;102(2):250-6). TG derived from either exogenous or endogenous sources isincorporated and secreted in chylomicrons from the intestine or in verylow density lipoproteins (VLDL) from the liver. Once in circulation, TGis hydrolyzed by lipoprotein lipase (LpL) and the resulting free fattyacids can then be taken up by local tissues and used as an energysource. Due to the profound effect LpL has on plasma TG and metabolismin general, discovering and developing compounds that affect LpLactivity are of great interest.

Metabolic syndrome is a combination of medical disorders that increaseone's risk for cardiovascular disease and diabetes. The symptoms,including high blood pressure, high triglycerides, decreased HDL andobesity, tend to appear together in some individuals. It affects a largenumber of people in a clustered fashion. In some studies, the prevalencein the USA is calculated as being up to 25% of the population. Metabolicsyndrome is known under various other names, such as (metabolic)syndrome X, insulin resistance syndrome, Reaven's syndrome or CHAOS.With the high prevalence of cardiovascular disorders and metabolicdisorders there remains a need for improved approaches to treat theseconditions

The angiopoietins are a family of secreted growth factors. Together withtheir respective endothelium-specific receptors, the angiopoietins playimportant roles in angiogenesis. One family member, angiopoietin-like 3(also known as ANGPT5, ANGPTL3, or angiopoietin 5), is predominantlyexpressed in the liver, and is thought to play a role in regulatinglipid metabolism (Kaplan et al., J. Lipid Res., 2003, 44, 136-143).

The human gene for angiopoietin-like 3 was identified and cloned as aresult of searches of assembled EST databases. The full-length humancDNA codes for a polypeptide of 460 amino acids which has thecharacteristic structural features of angiopoietins: a signal peptide,an extended helical domain, a short linker peptide, and a globularfibrinogen homology domain (FHD). The mouse angiopoietin-like 3 cDNA wasfound to encode a 455 amino acid polypeptide with 76% identity to thehuman polypeptide. An alignment of angiopoietins showed thatangiopoietin-like 3, unlike other family members, does not contain themotif of acidic residues determining a calcium binding site. Northernblot analysis revealed expression principally in the liver of adulttissues, with murine embryo Northern blots showing the presence oftranscripts as early as day 15, suggesting that angiopoietin-like 3 isexpressed early during liver development and that expression ismaintained in adult liver. The mouse gene maps to chromosome 4, and thehuman gene was mapped to the 1p31 region (Conklin et al., Genomics,1999, 62, 477-482).

KK obese mice have a multigenic syndrome of moderate obesity and adiabetic phenotype that resembles human hereditary type 2 diabetes.These mice show signs of hyperinsulinemia, hyperglycemia, andhyperlipidemia. A strain of KK mice called KK/San has significantly lowplasma lipid levels despite signs of hyperinsulinemia and hyperglycemia.The mutant phenotype is inherited recessively, and the locus was namedhypolipidemia (hypl). The locus maps to the middle of chromosome 4, andthe gene was identified as angiopoietin-like 3 through positionalcloning.

Injection of recombinant adenoviruses containing the full-length mouseor human angiopoietin-like 3 cDNA in the mutant KK/San mice caused anincrease in plasma levels of triglyceride, total cholesterol andnon-esterified fatty acids (NEFA). Similarly, injection of recombinantangiopoietin-like 3 protein into the mutant mice increased levels oftriglycerides and non-esterified fatty acids. (Koishi et al., Nat.Genet., 2002, 30, 151-157).

In another study focusing on the metabolic pathways of triglycerides inKK/San mice, overexpression of angiopoietin-like 3 resulted in a markedincrease of triglyceride-enriched very low density lipoportien (VLDL).Differences in the hepatic VLDL triglyceride secretion rate were notsignificant between wild-type KK and KK/San mice. However, studies withlabeled VLDL suggested that the low plasma triglyceride levels in KK/Sanmice were primarily due to enhanced lipolysis of VLDL triglyceridesrather than to enhanced whole particle uptake. The plasma apoB100 andapoB48 levels of KK/San mice were similar to wild-type KK mice.ApoCIII-deficient mice have a similar phenotype to KK/San mice, andApoCIII is thought to modulate VLDL triglyceride metabolism through theinhibition of lipase-mediated hydrolysis of VLDL triglycerides. In vitroanalysis of recombinant protein revealed that angiopoietin-like 3directly inhibits lipoprotein lipase (LPL) activity (Shimizugawa et al.,J. Biol. Chem., 2002, 277, 33742-33748).

Consistent with a role in lipid metabolism, angiopoietin-like 3 mRNA wasfound to be upregulated in C57BL/6J mice fed normal chow diets with 4%cholesterol and in mice treated with the liver X receptor (LXR) agonistT0901317. LXRs are ligand-activated transcription factors which play arole in the regulation of genes that govern cholesterol homeostasis inthe liver and peripheral tissues. In addition to cholesterol metabolism,LXRs may also play a role in regulation of fatty acid metabolism.Treatment of HepG2 cells with natural or synthetic agents which activateLXR caused increased angiopoietin-like 3 expression. The promoter of thehuman angiopoietin-like 3 gene was found to contain an LXR responseelement. In addition, the promoter contained several potential bindingsites for other transcription factors including HNF-1, HNF-4, and C/EBP.(Kaplan et al., J. Lipid Res., 2003, 44, 136-143).

Treatment of rodents with T0901317 is associated with triglycerideaccumulation in the liver and plasma. The liver triglycerideaccumulation has been explained by increased expression of the sterolregulatory element binding protein-1c (SREBP 1c) and fatty acid synthase(FAS), both of which are targets of LXR. T0901317 failed to increaseplasma triglyceride concentration in angiopoietin-like 3 deficient mice,while the stimulated accumulation of hepatic triglyceride was similar tothat observed in treated wild type mice. The rise in plasma triglyceridein wild-type mice treated with T0901317 parallels an induction ofangiopoietin-like 3 mRNA in the liver and an increase in plasmaconcentration of the protein. (Inaba et al., J. Biol. Chem., 2003, 278,21344-21351).

Further studies addressed the mechanism of the increase in plasma freefatty acid (FFA) levels observed in KK/Snk mice treated with exogenousangiopoietin-like 3. Probe of fixed human tissues with afluorescence-labeled angiopoietin-like 3 protein demonstrated strongbinding only on adipose tissue. Furthermore, radiolabeled proteinbinding was examined in 3T3-L1 adipocytes and was found to be saturableand specific. Incubation of 3T3-L1 adipocytes with angiopoietin-like 3led to enhanced release of FFA and glycerol into the culture medium.(Shimamura et al., Biochem. Biophys. Res. Commun., 2003, 301, 604-609).

In a study using streptozotocin-treated mice (STZ) to model theinsulin-deficient state and db/db mice to model the insulin-resistantdiabetic state, larger amounts of hepatic angiopoietin-like 3 wereobserved in diabetic mice as compared to control animals. Both models ofdiabetes showed hypertriglyceridemia, and the hyperlipidemia observedwas explained at least partially by the increased expression ofangiopoietin-like 3. These results suggested that angiopoietin-like 3 isa link between diabetes and dyslipidemia, with elevation promotinghyperlipidemia (Inukai et al., Biochem. Biophys. Res. Commun., 2004,317, 1075-1079).

A subsequent study examined the regulation of angiopoietin-like 3 byleptin and insulin, both of which are key players in the metabolicsyndrome. Angiopoietin-like 3 expression and plasma protein levels wereincreased in leptin-resistant db/db and leptin-deficient ob/ob micerelative to controls. Supplementation of ob/ob mice with leptindecreased angiopoietin-like 3 levels. The alterations in expression wereassociated with alterations in plasma triglyceride and free fatty acidlevels. Gene expression and plasma protein levels were also increased ininsulin-deficient STZ-treated mice. (Shimamura et al., Biochem BiophysRes Commun, 2004, 322, 1080-1085).

In accord with its membership in the angiopoietin family, recombinantangiopoietin-like 3 protein was found to bind to α_(V)β₃ integrin andinduced integrin α_(V)β₃-dependent haptotactic endothelial cell adhesionand migration. It also stimulated signal transduction pathwayscharacteristic for integrin activation. Angiopoietin-like 3 stronglyinduced angiogenesis in the rat corneal angiogenesis assay. (Camenischet al., J. Biol. Chem., 2002, 277, 17281-17290).

Genome-wide association scans (GWAS) surveying the genome for commonvariants associated with plasma concentrations of HDL, LDL andtriglyceride were undertaken by several groups. The GWAS studies foundan association between triglycerides and single-nucleotide polymorphisms(SNPs) near ANGPTL3 (Willer et al., Nature Genetics, 2008,40(2):161-169).

U.S. Pat. No. 7,267,819, application U.S. Ser. No. 12/128,545, andapplication U.S. Ser. No. 12/001,012 generally describeangiopoietin-like 3 agonists and antagonists.

PCT publications WO/02101039 (EP02733390) and WO/0142499 (U.S. Ser. No.10/164,030) disclose a nucleic acid sequence complementary to mouseangiopoietin-like 3 (Ryuta, 2002; Ryuta, 2001).

There is a currently a lack of acceptable options for treatingcardiovascular and metabolic disorders. It is therefore an object hereinto provide compounds and methods for the treatment of such diseases anddisorder.

The potential role of angiopoietin-like 3 in lipid metabolism makes itan attractive target for investigation. Antisense technology is emergingas an effective means for reducing the expression of certain geneproducts and may therefore prove to be uniquely useful in a number oftherapeutic, diagnostic, and research applications for the modulation ofangiopoietin-like 3.

SUMMARY OF THE INVENTION

Provided herein are antisense compounds useful for modulating geneexpression and associated pathways via antisense mechanisms of actionsuch as RNaseH, RNAi and dsRNA enzymes, as well as other antisensemechanisms based on target degradation or target occupancy.

Provided herein are methods, compounds, and compositions for inhibitingexpression of ANGPTL3 and treating, preventing, delaying or amelioratinga ANGPTL3 related disease, condition or a symptom thereof. In certainembodiments, the ANGPTL3 related disease or condition is cardiovasculardisease or metabolic disease.

In certain embodiments, the compounds or compositions of the inventioncomprise a modified oligonucleotide 10 to 30 linked nucleosides inlength targeted to ANGPTL3. The ANGPTL target can have a sequenceselected from any one of SEQ ID NOs: 1-5. The modified oligonucleotidetargeting ANGPTL3 can have a nucleobase sequence comprising at least 8contiguous nucleobases complementary to an equal length portion of SEQID NOs: 1-5. The modified oligonucleotide targeting ANGPTL3 can have anucleobase sequence comprising at least 8 contiguous nucleobases of anucleobase sequence selected from any of SEQ ID NO: 34-182. The modifiedoligonucleotide can have a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from a sequence recited in any one of SEQID NOs: 34-182. The contiguous nucleobase portion of the modifiedoligonucleotide can be complementary to an equal length portion of anANGPTL3 region selected from any one of SEQ ID NOs: 1-5. The ANGPTL3region can be chosen from one or more of the following regions: 22-52,116-145, 637-720, 953-983, 1333-1469 and 1463-1489.

Certain embodiments provide a method of reducing ANGPTL3 expression inan animal comprising administering to the animal a compound comprisingthe modified oligonucleotide targeting ANGPTL3 described herein.

Certain embodiments provide a method of reducing apoC-III expression,triglyceride levels, cholesterol levels, low-density lipoprotein (LDL)or glucose levels in an animal comprising administering to the animal acompound comprising the modified oligonucleotide targeted to ANGPTL3described herein, wherein the modified oligonucleotide reduces ANGPTL3expression in the animal.

Certain embodiments provide a method of ameliorating cardiovasculardisease or metabolic disease in an animal comprising administering tothe animal a compound comprising a modified oligonucleotide targeted toANGPTL3 described herein, wherein the modified oligonucleotide reducesANGPTL3 expression in the animal.

Certain embodiments provide a method for treating an animal withcardiovascular disease or metabolic disease comprising: 1) identifyingthe animal with cardiovascular disease or metabolic disease, and 2)administering to the animal a therapeutically effective amount of acompound comprising a modified oligonucleotide consisting of 20 linkednucleosides and having a nucleobase sequence at least 90% complementaryto SEQ ID NO: 1-5 as measured over the entirety of said modifiedoligonucleotide, thereby treating the animal with cardiovascular diseaseor metabolic disease. In certain embodiments, the therapeuticallyeffective amount of the compound administered to the animal reducescardiovascular disease or metabolic disease in the animal.

Certain embodiments provide a method for decreasing one or more ofANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels,cholesterol levels, glucose levels, fat pad weight, cardiovasculardisease and metabolic disease in a human by administering an ANGPTL3inhibitor comprising a modified oligonucleotide as described herein.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated-by-reference forthe portions of the document discussed herein, as well as in theirentirety.

DEFINITIONS

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques can be used for chemical synthesis, andchemical analysis. Where permitted, all patents, applications, publishedapplications and other publications, GENBANK Accession Numbers andassociated sequence information obtainable through databases such asNational Center for Biotechnology Information (NCBI) and other datareferred to throughout in the disclosure herein are incorporated byreference for the portions of the document discussed herein, as well asin their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to anO-methoxy-ethyl modification of the 2′ position of a furosyl ring. A2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-O-methoxyethyl nucleotide” means a nucleotide comprising a2′-O-methoxyethyl modified sugar moiety.

“3′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 3′-most nucleotide of a particular antisensecompound.

“5′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 5′-most nucleotide of a particular antisensecompound.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position. A 5-methylcytosine is a modifiednucleobase.

“About” means within ±10% of a value. For example, if it is stated, “amarker may be increased by about 50%”, it is implied that the marker maybe increased between 45%-55%

“Active pharmaceutical agent” means the substance or substances in apharmaceutical composition that provide a therapeutic benefit whenadministered to an individual. For example, in certain embodiments anantisense oligonucleotide targeted to ANGPTL3 is an activepharmaceutical agent.

“Active target region” or “target region” means a region to which one ormore active antisense compounds is targeted.

“Active antisense compounds” means antisense compounds that reducetarget nucleic acid levels or protein levels.

“Adipogenesis” means the development of fat cells from preadipocytes.“Lipogenesis” means the production or formation of fat, either fattydegeneration or fatty infiltration.

“Adiposity” or “Obesity” refers to the state of being obese or anexcessively high amount of body fat or adipose tissue in relation tolean body mass. The amount of body fat includes concern for both thedistribution of fat throughout the body and the size and mass of theadipose tissue deposits. Body fat distribution can be estimated byskin-fold measures, waist-to-hip circumference ratios, or techniquessuch as ultrasound, computed tomography, or magnetic resonance imaging.According to the Center for Disease Control and Prevention, individualswith a body mass index (BMI) of 30 or more are considered obese. Theterm “Obesity” as used herein includes conditions where there is anincrease in body fat beyond the physical requirement as a result ofexcess accumulation of adipose tissue in the body. The term “obesity”includes, but is not limited to, the following conditions: adult-onsetobesity; alimentary obesity; endogenous or metabolic obesity; endocrineobesity; familial obesity; hyperinsulinar obesity;hyperplastic-hypertrophic obesity; hypogonadal obesity; hypothyroidobesity; lifelong obesity; morbid obesity and exogenous obesity.

“Administered concomitantly” refers to the co-administration of twoagents in any manner in which the pharmacological effects of both aremanifest in the patient at the same time. Concomitant administrationdoes not require that both agents be administered in a singlepharmaceutical composition, in the same dosage form, or by the sameroute of administration. The effects of both agents need not manifestthemselves at the same time. The effects need only be overlapping for aperiod of time and need not be coextensive.

“Administering” means providing an agent to an animal, and includes, butis not limited to, administering by a medical professional andself-administering.

“Agent” means an active substance that can provide a therapeutic benefitwhen administered to an animal. “First Agent” means a therapeuticcompound of the invention. For example, a first agent can be anantisense oligonucleotide targeting ANGPTL3. “Second agent” means asecond therapeutic compound of the invention (e.g. a second antisenseoligonucleotide targeting ANGPTL3) and/or a non-ANGPTL3 therapeuticcompound.

“Amelioration” refers to a lessening of at least one indicator, sign, orsymptom of an associated disease, disorder, or condition. The severityof indicators can be determined by subjective or objective measures,which are known to those skilled in the art.

“ANGPTL3” means any nucleic acid or protein of ANGPTL3.

“ANGPTL3 expression” means the level of mRNA transcribed from the geneencoding ANGPTL3 or the level of protein translated from the mRNA.ANGPTL3 expression can be determined by art known methods such as aNorthern or Western blot.

“ANGPTL3 nucleic acid” means any nucleic acid encoding ANGPTL3. Forexample, in certain embodiments, a ANGPTL3 nucleic acid includes a DNAsequence encoding ANGPTL3, a RNA sequence transcribed from DNA encodingANGPTL3 (including genomic DNA comprising introns and exons), and a mRNAsequence encoding ANGPTL3. “ANGPTL3 mRNA” means a mRNA encoding anANGPTL3 protein.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding.

“Antisense inhibition” means reduction of target nucleic acid levels ortarget protein levels in the presence of an antisense compoundcomplementary to a target nucleic acid compared to target nucleic acidlevels or target protein levels in the absence of the antisensecompound.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding region or segment of a target nucleic acid.

“ApoB-containing lipoprotein” means any lipoprotein that hasapolipoprotein B as its protein component, and is understood to includeLDL, VLDL, IDL, and lipoprotein(a) and can be generally targeted bylipid lowering agent and therapies. “ApoB-100-containing LDL” meansApoB-100 isoform containing LDL.

“Atherosclerosis” means a hardening of the arteries affecting large andmedium-sized arteries and is characterized by the presence of fattydeposits. The fatty deposits are called “atheromas” or “plaques,” whichconsist mainly of cholesterol and other fats, calcium and scar tissue,and damage the lining of arteries.

“Bicyclic sugar” means a furosyl ring modified by the bridging of twonon-geminal ring atoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotidewherein the furanose portion of the nucleoside or nucleotide includes abridge connecting two carbon atoms on the furanose ring, thereby forminga bicyclic ring system.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“Cardiovascular disease” or “cardiovascular disorder” refers to a groupof conditions related to the heart, blood vessels, or the circulation.Examples of cardiovascular diseases or disorders include, but are notlimited to, aneurysm, angina, arrhythmia, atherosclerosis,cerebrovascular disease (stroke), coronary heart disease, hypertension,dyslipidemia, hyperlipidemia, and hypercholesterolemia.

“Chemically distinct region” refers to a region of an antisense compoundthat is in some way chemically different than another region of the sameantisense compound. For example, a region having 2′-O-methoxyethylnucleotides is chemically distinct from a region having nucleotideswithout 2′-O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has atleast two chemically distinct regions.

“Co-administration” means administration of two or more agents to anindividual. The two or more agents can be in a single pharmaceuticalcomposition, or can be in separate pharmaceutical compositions. Each ofthe two or more agents can be administered through the same or differentroutes of administration. Co-administration encompasses parallel orsequential administration.

“Cholesterol” is a sterol molecule found in the cell membranes of allanimal tissues. Cholesterol must be transported in an animal's bloodplasma by lipoproteins including very low density lipoprotein (VLDL),intermediate density lipoprotein (IDL), low density lipoprotein (LDL),and high density lipoprotein (HDL). “Plasma cholesterol” refers to thesum of all lipoproteins (VDL, IDL, LDL, HDL) esterified and/ornon-estrified cholesterol present in the plasma or serum.

“Cholesterol absorption inhibitor” means an agent that inhibits theabsorption of exogenous cholesterol obtained from diet.

“Complementarity” means the capacity for pairing between nucleobases ofa first nucleic acid and a second nucleic acid. In certain embodiments,complementarity between the first and second nucleic acid may be betweentwo DNA strands, between two RNA strands, or between a DNA and an RNAstrand. In certain embodiments, some of the nucleobases on one strandare matched to a complementary hydrogen bonding base on the otherstrand. In certain embodiments, all of the nucleobases on one strand arematched to a complementary hydrogen bonding base on the other strand. Incertain embodiments, a first nucleic acid is an antisense compound and asecond nucleic acid is a target nucleic acid. In certain suchembodiments, an antisense oligonucleotide is a first nucleic acid and atarget nucleic acid is a second nucleic acid.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Coronary heart disease (CHD)” means a narrowing of the small bloodvessels that supply blood and oxygen to the heart, which is often aresult of atherosclerosis.

“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′position of the sugar portion of the nucleotide. Deoxyribonucleotidesmay be modified with any of a variety of substituents.

“Diabetes mellitus” or “diabetes” is a syndrome characterized bydisordered metabolism and abnormally high blood sugar (hyperglycemia)resulting from insufficient levels of insulin or reduced insulinsensitivity. The characteristic symptoms are excessive urine production(polyuria) due to high blood glucose levels, excessive thirst andincreased fluid intake (polydipsia) attempting to compensate forincreased urination, blurred vision due to high blood glucose effects onthe eye's optics, unexplained weight loss, and lethargy.

“Diabetic dyslipidemia” or “type 2 diabetes with dyslipidemia” means acondition characterized by Type 2 diabetes, reduced HDL-C, elevatedtriglycerides, and elevated small, dense LDL particles.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, the diluent in an injected composition can be aliquid, e.g. saline solution.

“Dyslipidemia” refers to a disorder of lipid and/or lipoproteinmetabolism, including lipid and/or lipoprotein overproduction ordeficiency. Dyslipidemias may be manifested by elevation of lipids suchas cholesterol and triglycerides as well as lipoproteins such aslow-density lipoprotein (LDL) cholesterol.

“Dosage unit” means a form in which a pharmaceutical agent is provided,e.g. pill, tablet, or other dosage unit known in the art. In certainembodiments, a dosage unit is a vial containing lyophilized antisenseoligonucleotide. In certain embodiments, a dosage unit is a vialcontaining reconstituted antisense oligonucleotide.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose can be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections can be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses can be stated as theamount of pharmaceutical agent per hour, day, week, or month. Doses canbe expressed as mg/kg or g/kg.

“Effective amount” or “therapeutically effective amount” means theamount of active pharmaceutical agent sufficient to effectuate a desiredphysiological outcome in an individual in need of the agent. Theeffective amount can vary among individuals depending on the health andphysical condition of the individual to be treated, the taxonomic groupof the individuals to be treated, the formulation of the composition,assessment of the individual's medical condition, and other relevantfactors.

“Fully complementary” or “100% complementary” means each nucleobase of anucleobase sequence of a first nucleic acid has a complementarynucleobase in a second nucleobase sequence of a second nucleic acid. Incertain embodiments, a first nucleic acid is an antisense compound and atarget nucleic acid is a second nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal regionhaving a plurality of nucleosides that support RNase H cleavage ispositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region can be referred to as a “gap segment” andthe external regions can be referred to as “wing segments.”

“Gap-widened” means a chimeric antisense compound having a gap segmentof 12 or more contiguous 2′-deoxyribonucleosides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from one to sixnucleosides.

“Glucose” is a monosaccharide used by cells as a source of energy andmetabolic intermediate. “Plasma glucose” refers to glucose present inthe plasma.

“High density lipoprotein-C(HDL-C)” means cholesterol associated withhigh density lipoprotein particles. Concentration of HDL-C in serum (orplasma) is typically quantified in mg/dL or nmol/L. “serum HDL-C” and“plasma HDL-C” mean HDL-C in serum and plasma, respectively.

“HMG-CoA reductase inhibitor” means an agent that acts through theinhibition of the enzyme HMG-CoA reductase, such as atorvastatin,rosuvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin.

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude an antisense compound and a target nucleic acid.

“Hypercholesterolemia” means a condition characterized by elevatedcholesterol or circulating (plasma) cholesterol, LDL-cholesterol andVLDL-cholesterol, as per the guidelines of the Expert Panel Report ofthe National Cholesterol Educational Program (NCEP) of Detection,Evaluation of Treatment of high cholesterol in adults (see, Arch. Int.Med. (1988) 148, 36-39).

“Hyperlipidemia” or “hyperlipemia” is a condition characterized byelevated serum lipids or circulating (plasma) lipids. This conditionmanifests an abnormally high concentration of fats. The lipid fractionsin the circulating blood are cholesterol, low density lipoproteins, verylow density lipoproteins and triglycerides.

“Hypertriglyceridemia” means a condition characterized by elevatedtriglyceride levels.

“Identifying” or “selecting a subject having a metabolic orcardiovascular disease” means identifying or selecting a subject havingbeen diagnosed with a metabolic disease, a cardiovascular disease, or ametabolic syndrome; or, identifying or selecting a subject having anysymptom of a metabolic disease, cardiovascular disease, or metabolicsyndrome including, but not limited to, hypercholesterolemia,hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertensionincreased insulin resistance, decreased insulin sensitivity, abovenormal body weight, and/or above normal body fat content or anycombination thereof. Such identification may be accomplished by anymethod, including but not limited to, standard clinical tests orassessments, such as measuring serum or circulating (plasma)cholesterol, measuring serum or circulating (plasma) blood-glucose,measuring serum or circulating (plasma) triglycerides, measuringblood-pressure, measuring body fat content, measuring body weight, andthe like.

“Identifying” or “selecting a diabetic subject” means identifying orselecting a subject having been identified as diabetic or identifying orselecting a subject having any symptom of diabetes (type 1 or type 2)such as, but not limited to, having a fasting glucose of at least 110mg/dL, glycosuria, polyuria, polydipsia, increased insulin resistance,and/or decreased insulin sensitivity.

“Identifying” or “selecting an obese subject” means identifying orselecting a subject having been diagnosed as obese or identifying orselecting a subject with a BMI over 30 and/or a waist circumference ofgreater than 102 cm in men or greater than 88 cm in women.

“Identifying” or “selecting a subject having dyslipidemia” meansidentifying or selecting a subject diagnosed with a disorder of lipidand/or lipoprotein metabolism, including lipid and/or lipoproteinoverproduction or deficiency. Dyslipidemias may be manifested byelevation of lipids such as cholesterol and triglycerides as well aslipoproteins such as low-density lipoprotein (LDL) cholesterol.

“Identifying” or “selecting” a subject having increased adiposity” meansidentifying or selecting a subject having an increased amount of bodyfat (or adiposity) that includes concern for one or both thedistribution of fat throughout the body and the size and mass of theadipose tissue deposits. Body fat distribution can be estimated byskin-fold measures, waist-to-hip circumference ratios, or techniquessuch as ultrasound, computer tomography, or magnetic resonance imaging.According to the Center for Disease Control and Prevention, individualswith a body mass index (BMI) of 30 or more are considered obese.

“Improved cardiovascular outcome” means a reduction in the occurrence ofadverse cardiovascular events, or the risk thereof. Examples of adversecardiovascular events include, without limitation, death, reinfarction,stroke, cardiogenic shock, pulmonary edema, cardiac arrest, and atrialdysrhythmia.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements.

“Individual” or “subject” or “animal” means a human or non-human animalselected for treatment or therapy.

“Inhibiting the expression or activity” refers to a reduction orblockade of the expression or activity and does not necessarily indicatea total elimination of expression or activity.

“Insulin resistance” is defined as the condition in which normal amountsof insulin are inadequate to produce a normal insulin response fromcells, e.g., fat, muscle and/or liver cells. Insulin resistance in fatcells results in hydrolysis of stored triglycerides, which elevates freefatty acids in the blood plasma. Insulin resistance in muscle reducesglucose uptake whereas insulin resistance in liver reduces glucosestorage, with both effects serving to elevate blood glucose. High plasmalevels of insulin and glucose due to insulin resistance often leads tometabolic syndrome and type 2 diabetes.

“Insulin sensitivity” is a measure of how effectively an individualprocesses glucose. An individual having high insulin sensitivityeffectively processes glucose whereas an individual with low insulinsensitivity does not effectively process glucose.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“Intravenous administration” means administration into a vein.

“Linked nucleosides” means adjacent nucleosides which are bondedtogether.

“Lipid-lowering” means a reduction in one or more lipids in a subject.Lipid-lowering can occur with one or more doses over time.

“Lipid-lowering agent” means an agent, for example, an ANGPTL3-specificmodulator, provided to a subject to achieve a lowering of lipids in thesubject. For example, in certain embodiments, a lipid-lowering agent isprovided to a subject to reduce one or more of apoB, apoC3, totalcholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small denseLDL particles, and Lp(a) in a subject. “Lipid-lowering therapy” means atherapeutic regimen provided to a subject to reduce one or more lipidsin a subject. In certain embodiments, a lipid-lowering therapy isprovided to reduce one or more of apoB, apoC-III, total cholesterol,LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small dense LDLparticles, and Lp(a) in a subject.

“Lipoprotein”, such as VLDL, LDL and HDL, refers to a group of proteinsfound in the serum, plasma and lymph and are important for lipidtransport. The chemical composition of each lipoprotein differs in thatthe HDL has a higher proportion of protein versus lipid, whereas theVLDL has a lower proportion of protein versus lipid.

“Low density lipoprotein-cholesterol (LDL-C)” means cholesterol carriedin low density lipoprotein particles. Concentration of LDL-C in serum(or plasma) is typically quantified in mg/dL or nmol/L. “Serum LDL-C”and “plasma LDL-C” mean LDL-C in the serum and plasma, respectively.

“Major risk factors” refers to factors that contribute to a high riskfor a particular disease or condition. In certain embodiments, majorrisk factors for coronary heart disease include, without limitation,cigarette smoking, hypertension, low HDL-C, family history of coronaryheart disease, age, and other factors disclosed herein.

“Metabolic disorder” or “metabolic disease” refers to a conditioncharacterized by an alteration or disturbance in metabolic function.“Metabolic” and “metabolism” are terms well known in the art andgenerally include the whole range of biochemical processes that occurwithin a living organism. Metabolic disorders include, but are notlimited to, hyperglycemia, prediabetes, diabetes (type I and type 2),obesity, insulin resistance, metabolic syndrome and dyslipidemia due totype 2 diabetes.

“Metabolic syndrome” means a condition characterized by a clustering oflipid and non-lipid cardiovascular risk factors of metabolic origin. Incertain embodiments, metabolic syndrome is identified by the presence ofany 3 of the following factors: waist circumference of greater than 102cm in men or greater than 88 cm in women; serum triglyceride of at least150 mg/dL; HDL-C less than 40 mg/dL in men or less than 50 mg/dL inwomen; blood pressure of at least 130/85 mmHg; and fasting glucose of atleast 110 mg/dL. These determinants can be readily measured in clinicalpractice (JAMA, 2001, 285: 2486-2497).

“Mismatch” or “non-complementary nucleobase” refers to the case when anucleobase of a first nucleic acid is not capable of pairing with thecorresponding nucleobase of a second or target nucleic acid.

“Mixed dyslipidemia” means a condition characterized by elevatedcholesterol and elevated triglycerides.

“Modified internucleoside linkage” refers to a substitution or anychange from a naturally occurring internucleoside bond (i.e. aphosphodiester internucleoside bond).

“Modified nucleobase” refers to any nucleobase other than adenine,cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase”means the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C), and uracil (U).

“Modified nucleoside” means a nucleoside having, independently, one ormore modified sugar moiety or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, one ormore modified sugar moiety, modified internucleoside linkage, ormodified nucleobase. A “modified nucleoside” means a nucleoside having,independently, one or more modified sugar moiety or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at leastone modified nucleotide.

“Modified sugar” refers to a substitution or change from a naturalsugar.

“Motif” means the pattern of chemically distinct regions in an antisensecompound.

“MTP inhibitor” means an agent inhibits the enzyme microsomaltriglyceride transfer protein.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Natural sugar moiety” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Non-alcoholic fatty liver disease” or “NAFLD” means a conditioncharacterized by fatty inflammation of the liver that is not due toexcessive alcohol use (for example, alcohol consumption of over 20g/day). In certain embodiments, NAFLD is related to insulin resistanceand metabolic syndrome. NAFLD encompasses a disease spectrum rangingfrom simple triglyceride accumulation in hepatocytes (hepatic steatosis)to hepatic steatosis with inflammation (steatohepatitis), fibrosis, andcirrhosis.

“Nonalcoholic steatohepatitis” (NASH) occurs from progression of NAFLDbeyond deposition of triglycerides. A “second hit” capable of inducingnecrosis, inflammation, and fibrosis is required for development ofNASH. Candidates for the second-hit can be grouped into broadcategories: factors causing an increase in oxidative stress and factorspromoting expression of proinflammatory cytokines. It has been suggestedthat increased liver triglycerides lead to increased oxidative stress inhepatocytes of animals and humans, indicating a potentialcause-and-effect relationship between hepatic triglyceride accumulation,oxidative stress, and the progression of hepatic steatosis to NASH(Browning and Horton, J Clin Invest, 2004, 114, 147-152).Hypertriglyceridemia and hyperfattyacidemia can cause triglycerideaccumulation in peripheral tissues (Shimamura et al., Biochem BiophysRes Commun, 2004, 322, 1080-1085).

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids(DNA), single-stranded nucleic acids, double-stranded nucleic acids,small interfering ribonucleic acids (siRNA), and microRNAs (miRNA). Anucleic acid can also comprise a combination of these elements in asingle molecule.

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugaror the sugar and the base and not necessarily the linkage at one or morepositions of an oligomeric compound such as for example nucleosidemimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl,bicyclo or tricyclo sugar mimetics e.g. non furanose sugar units.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside.

“Nucleotide mimetic” includes those structures used to replace thenucleoside and the linkage at one or more positions of an oligomericcompound such as for example peptide nucleic acids or morpholinos(morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiesterlinkage).

“Oligomeric compound” or “oligomer” refers to a polymeric structurecomprising two or more sub-structures and capable of hybridizing to aregion of a nucleic acid molecule. In certain embodiments, oligomericcompounds are oligonucleosides. In certain embodiments, oligomericcompounds are oligonucleotides. In certain embodiments, oligomericcompounds are antisense compounds. In certain embodiments, oligomericcompounds are antisense oligonucleotides. In certain embodiments,oligomeric compounds are chimeric oligonucleotides.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another.

“Parenteral administration” means administration by a manner other thanthrough the digestive tract. Parenteral administration includes topicaladministration, subcutaneous administration, intravenous administration,intramuscular administration, intraarterial administration,intraperitoneal administration, or intracranial administration, e.g.intrathecal or intracerebroventricular administration. Administrationcan be continuous, or chronic, or short or intermittent.

“Peptide” means a molecule formed by linking at least two amino acids byamide bonds. Peptide refers to polypeptides and proteins.

“Pharmaceutical agent” means a substance that provides a therapeuticbenefit when administered to an individual. For example, in certainembodiments, an antisense oligonucleotide targeted to ANGPTL3 ispharmaceutical agent.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual. For example, a pharmaceuticalcomposition can comprise one or more active agents and a sterile aqueoussolution.

“Pharmaceutically acceptable carrier” means a medium or diluent thatdoes not interfere with the structure or function of theoligonucleotide. Certain, of such carries enable pharmaceuticalcompositions to be formulated as, for example, tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspension and lozenges forthe oral ingestion by a subject. Certain of such carriers enablepharmaceutical compositions to be formulated for injection or infusion.For example, a pharmaceutically acceptable carrier can be a sterileaqueous solution.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage is amodified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e. linked) nucleobasesof a nucleic acid. In certain embodiments, a portion is a defined numberof contiguous nucleobases of a target nucleic acid. In certainembodiments, a portion is a defined number of contiguous nucleobases ofan antisense compound.

“Prevent” refers to delaying or forestalling the onset or development ofa disease, disorder, or condition for a period of time from minutes toindefinitely. Prevent also means reducing risk of developing a disease,disorder, or condition.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form within the body or cells thereof bythe action of endogenous enzymes or other chemicals or conditions.

“Side effects” means physiological responses attributable to a treatmentother than the desired effects. In certain embodiments, side effectsinclude injection site reactions, liver function test abnormalities,renal function abnormalities, liver toxicity, renal toxicity, centralnervous system abnormalities, myopathies, and malaise. For example,increased aminotransferase levels in serum can indicate liver toxicityor liver function abnormality. For example, increased bilirubin canindicate liver toxicity or liver function abnormality.

“Single-stranded oligonucleotide” means an oligonucleotide which is nothybridized to a complementary strand.

“Specifically hybridizable” refers to an antisense compound having asufficient degree of complementarity with a target nucleic acid toinduce a desired effect, while exhibiting minimal or no effects onnon-target nucleic acids under conditions in which specific binding isdesired, i.e. under physiological conditions in the case of in vivoassays and therapeutic treatments.

“Statin” means an agent that inhibits the activity of HMG-CoA reductase.

“Subcutaneous administration” means administration just below the skin.

“Targeting” or “targeted” means the process of design and selection ofan antisense compound that will specifically hybridize to a targetnucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” allrefer to a nucleic acid capable of being targeted by antisensecompounds.

“Target region” is defined as a portion of the target nucleic acidhaving at least one identifiable structure, function, or characteristic.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which one or more antisense compound is targeted. “5′ targetsite” refers to the 5′-most nucleotide of a target segment. “3′ targetsite” refers to the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of an agent thatprovides a therapeutic benefit to an individual.

“Therapeutic lifestyle change” means dietary and lifestyle changesintended to lower fat/adipose tissue mass and/or cholesterol. Suchchange can reduce the risk of developing heart disease, and may includerecommendations for dietary intake of total daily calories, total fat,saturated fat, polyunsaturated fat, monounsaturated fat, carbohydrate,protein, cholesterol, insoluble fiber, as well as recommendations forphysical activity.

“Triglyceride” means a lipid or neutral fat consisting of glycerolcombined with three fatty acid molecules.

“Type 2 diabetes,” (also known as “type 2 diabetes mellitus” or“diabetes mellitus, type 2”, and formerly called “diabetes mellitus type2”, “non-insulin-dependent diabetes (NIDDM)”, “obesity relateddiabetes”, or “adult-onset diabetes”) is a metabolic disorder that isprimarily characterized by insulin resistance, relative insulindeficiency, and hyperglycemia.

“Treat” refers to administering a pharmaceutical composition to effectan alteration or improvement of a disease, disorder, or condition.

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties, and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is a RNA nucleotide (i.e.β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

Certain Embodiments

In certain embodiments, the compounds or compositions of the inventioncomprise a modified oligonucleotide 10 to 30 linked nucleosides inlength targeted to ANGPTL3. The ANGPTL target can have a sequenceselected from any one of SEQ ID NOs: 1-5.

In certain embodiments, the compounds or compositions of the inventioncomprise a modified oligonucleotide consisting of 10 to 30 nucleosideshaving a nucleobase sequence comprising at least 8 contiguousnucleobases complementary to an equal length portion of SEQ ID NOs: 1-5.

In certain embodiments, the compounds or compositions of the inventioncomprise a modified oligonucleotide consisting of 10 to 30 nucleosideshaving a nucleobase sequence comprising at least 8 contiguousnucleobases of a nucleobase sequence selected from any of SEQ ID NO:34-182.

In certain embodiments, the compounds or compositions of the inventioncomprise a modified oligonucleotide consisting of 10 to 30 linkednucleosides and having a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from a sequence recited in any one of SEQID NOs: 34-182.

In certain embodiments, the compounds or compositions of the inventioncomprise a salt of the modified oligonucleotide.

In certain embodiments, the compounds or compositions of the inventionfurther comprise a pharmaceutically acceptable carrier or diluent.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is at least 70%, 80%, 90%, 95% or 100% complementary toany one of SEQ ID NO: 1-5 as measured over the entirety of the modifiedoligonucleotide.

In certain embodiments, the compound of the invention consists of asingle-stranded modified oligonucleotide.

In certain embodiments, the modified oligonucleotide consists of 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 linked nucleosides. In certain embodiments, the modifiedoligonucleotide consists of 20 linked nucleosides.

In certain embodiments, at least one internucleoside linkage of saidmodified oligonucleotide is a modified internucleoside linkage. Incertain embodiments, each internucleoside linkage is a phosphorothioateinternucleoside linkage.

In certain embodiments, at least one nucleoside of the modifiedoligonucleotide comprises a modified sugar. In certain embodiments themodified oligonucleotide comprises at least one tetrahydropyran modifiednucleoside wherein a tetrahydropyran ring replaces a furanose ring. Incertain embodiments each of the tetrahydropyran modified nucleoside hasthe structure:

wherein Bx is an optionally protected heterocyclic base moiety. Incertain embodiments, at least one modified sugar is a bicyclic sugar. Incertain embodiments, at least one modified sugar comprises a2′-O-methoxyethyl or a 4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or 2.

In certain embodiments, at least one nucleoside of said modifiedoligonucleotide comprises a modified nucleobase. In certain embodiments,the modified nucleobase is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide comprises: a) a gapsegment consisting of linked deoxynucleosides; b) a 5′ wing segmentconsisting of linked nucleosides; and c) a 3′ wing segment consisting oflinked nucleosides. The gap segment is positioned between the 5′ wingsegment and the 3′ wing segment and each nucleoside of each wing segmentcomprises a modified sugar. In certain embodiments, the modifiedoligonucleotide consists of 20 linked nucleosides, the gap segmentconsisting of ten linked deoxynucleosides, the 5′ wing segmentconsisting of five linked nucleosides, the 3′ wing segment consisting offive linked nucleosides, each nucleoside of each wing segment comprisesa 2′-O-methoxyethyl sugar and each internucleoside linkage is aphosphorothioate linkage.

In certain embodiments, the compounds or compositions of the inventioncomprise a modified oligonucleotide consisting of 20 linked nucleosideshaving a nucleobase sequence comprising at least 8 contiguousnucleobases complementary to an equal length portion of SEQ ID NO: 1-5,wherein the modified oligonucleotide comprises: a) a gap segmentconsisting of ten linked deoxynucleosides; b) a 5′ wing segmentconsisting of five linked nucleosides; and c) a 3′ wing segmentconsisting of five linked nucleosides. The gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment, each nucleoside ofeach wing segment comprises a 2′-O-methoxyethyl sugar, eachinternucleoside linkage is a phosphorothioate linkage and each cytosineresidue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions of the inventioncomprise a modified oligonucleotide consisting of 20 linked nucleosideshaving a nucleobase sequence comprising at least 8 contiguousnucleobases of a nucleobase sequence selected from any of SEQ ID NO:34-182, wherein the modified oligonucleotide comprises: a) a gap segmentconsisting of ten linked deoxynucleosides; b) a 5′ wing segmentconsisting of five linked nucleosides; and c) a 3′ wing segmentconsisting of five linked nucleosides. The gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment, each nucleoside ofeach wing segment comprises a 2′-O-methoxyethyl sugar, eachinternucleoside linkage is a phosphorothioate linkage and each cytosineresidue is a 5-methylcytosine.

Certain embodiments provide methods, compounds, and compositions forinhibiting ANGPTL3 expression.

Certain embodiments provide a method of reducing ANGPTL3 expression inan animal comprising administering to the animal a compound comprising amodified oligonucleotide 10 to 30 linked nucleosides in length targetedto ANGPTL3.

Certain embodiments provide a method of reducing ApoC-III expression inan animal comprising administering to the animal a compound comprising amodified oligonucleotide 10 to 30 linked nucleosides in length targetedto ANGPTL3, thereby reducing the expression of ApoC-III in the animal.

Certain embodiments provide a method of reducing triglyceride levels inan animal comprising administering to the animal a compound comprising amodified oligonucleotide 10 to 30 linked nucleosides in length targetedto ANGPTL3, thereby reducing the level of triglyceride in the animal.

Certain embodiments provide a method of reducing cholesterol levels inan animal comprising administering to the animal a compound comprising amodified oligonucleotide 10 to 30 linked nucleosides in length targetedto ANGPTL3, thereby reducing the level of cholesterol in the animal.

Certain embodiments provide a method of reducing low-density lipoprotein(LDL) levels in an animal comprising administering to the animal acompound comprising a modified oligonucleotide 10 to 30 linkednucleosides in length targeted to ANGPTL3, thereby reducing the level oflow-density lipoprotein (LDL) in the animal.

Certain embodiments provide a method of reducing glucose levels in ananimal comprising administering to the animal a compound comprising amodified oligonucleotide 10 to 30 linked nucleosides in length targetedto ANGPTL3, thereby reducing the level of glucose in the animal.

Certain embodiments provide a method of ameliorating metabolic orcardiovascular disease in an animal comprising administering to theanimal a compound comprising a modified oligonucleotide 10 to 30 linkednucleosides in length targeted to ANGPTL3, thereby ameliorating themetabolic or cardiovascular disease in the animal.

Certain embodiments provide a method for treating an animal with anANGPTL3 related disease or condition comprising: a) identifying saidanimal with the ANGPTL3 related disease or condition, and b)administering to said animal a therapeutically effective amount of acompound comprising a modified oligonucleotide 10 to 30 linkednucleosides in length targeted to ANGPTL3. In certain embodiments, thetherapeutically effective amount of the compound administered to theanimal reduces the ANGPTL3 related disease or condition in the animal.

Certain embodiments provide a method for treating an animal withmetabolic or cardiovascular disease comprising: a) identifying saidanimal with metabolic or cardiovascular disease, and b) administering tosaid animal a therapeutically effective amount of a compound comprisinga modified oligonucleotide consisting of 20 linked nucleosides andhaving a nucleobase sequence at least 90% complementary to SEQ ID NO:1-5 as measured over the entirety of said modified oligonucleotide,thereby treating the animal with metabolic or cardiovascular disease. Incertain embodiments, the therapeutically effective amount of thecompound administered to the animal reduces the metabolic orcardiovascular disease in the animal.

Certain embodiments provide a method of decreasing one or more ofANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels,cholesterol levels, glucose levels, fat pad weight, cardiovasculardisease and metabolic disease in a human by administering an ANGPTL3inhibitor comprising a modified oligonucleotide consisting of 20 linkednucleosides and having a nucleobase sequence at least 90% complementaryto SEQ ID NO: 1-5 as measured over the entirety of said modifiedoligonucleotide.

Certain embodiments provide uses of the compounds and compositionsdescribed herein for inhibiting ANGPTL3 expression.

Certain embodiments provide use of the compounds and compositionsdescribed herein for reducing ANGPTL3 expression in an animal. Certainembodiments include administering to the animal a compound comprising amodified oligonucleotide 10 to 30 linked nucleosides in length targetedto ANGPTL3.

Certain embodiments provide use of the compounds and compositionsdescribed herein for reducing ApoC-III expression in an animal. Certainembodiments include administering to the animal a compound comprising amodified oligonucleotide 10 to 30 linked nucleosides in length targetedto ANGPTL3, thereby reducing the expression of ApoC-III in the animal.

Certain embodiments provide use of the compounds and compositionsdescribed herein for reducing triglyceride levels in an animal. Certainembodiments include administering to the animal a compound comprising amodified oligonucleotide 10 to 30 linked nucleosides in length targetedto ANGPTL3, thereby reducing the level of triglyceride in the animal.

Certain embodiments provide use of the compounds and compositionsdescribed herein for reducing cholesterol levels in an animal. Certainembodiments include administering to the animal a compound comprising amodified oligonucleotide 10 to 30 linked nucleosides in length targetedto ANGPTL3, thereby reducing the level of cholesterol in the animal.

Certain embodiments provide use of the compounds and compositionsdescribed herein for reducing low-density lipoprotein (LDL) levels in ananimal. Certain embodiments include administering to the animal acompound comprising a modified oligonucleotide 10 to 30 linkednucleosides in length targeted to ANGPTL3, thereby reducing the level oflow-density lipoprotein (LDL) in the animal.

Certain embodiments provide use of the compounds and compositionsdescribed herein for reducing glucose levels in an animal. Certainembodiments include administering to the animal a compound comprising amodified oligonucleotide 10 to 30 linked nucleosides in length targetedto ANGPTL3, thereby reducing the level of glucose in the animal.

Certain embodiments provide use of the compounds and compositionsdescribed herein for ameliorating metabolic or cardiovascular disease inan animal. Certain embodiments include administering to the animal acompound comprising a modified oligonucleotide 10 to 30 linkednucleosides in length targeted to ANGPTL3, thereby ameliorating themetabolic or cardiovascular disease in the animal.

Certain embodiments provide use of the compounds and compositionsdescribed herein for treatment. Certain embodiments provide use of thecompounds and compositions described herein for treating an animal withan ANGPTL3 related disease or condition. In certain embodiments, theANGPTL3 related disease or condition is metabolic or cardiovasculardisease. Certain embodiments include: a) identifying said animal withthe ANGPTL3 related disease or condition, and b) administering to saidanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide 10 to 30 linked nucleosides in length targetedto ANGPTL3. In certain embodiments, the therapeutically effective amountof the compound administered to the animal reduces the ANGPTL3 relateddisease or condition in the animal.

Certain embodiments provide use of the compounds and compositionsdescribed herein for treating an animal with metabolic or cardiovasculardisease. comprising: a) identifying said animal with metabolic orcardiovascular disease, and b) administering to said animal atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 20 linked nucleosides and having anucleobase sequence at least 90% complementary to SEQ ID NO: 1-5 asmeasured over the entirety of said modified oligonucleotide, therebytreating the animal with metabolic or cardiovascular disease. In certainembodiments, the therapeutically effective amount of the compoundadministered to the animal reduces the metabolic or cardiovasculardisease in the animal.

Certain embodiments provide use of the compounds and compositionsdescribed herein for decreasing one or more of ANGPTL3 levels, LDLlevels, apoC-III levels, triglyceride levels, cholesterol levels,glucose levels, fat pad weight, cardiovascular disease and metabolicdisease in a human by administering an ANGPTL3 inhibitor comprising amodified oligonucleotide consisting of 20 linked nucleosides and havinga nucleobase sequence at least 90% complementary to SEQ ID NO: 1-5 asmeasured over the entirety of said modified oligonucleotide.

In certain embodiments, ANGPTL3 has the sequence as set forth in GenBankAccession No. BG400407.1 (incorporated herein as SEQ ID NO: 1). Incertain embodiments, ANGPTL3 has the sequence as set forth in GenBankAccession No. BG562555.1 (incorporated herein as SEQ ID NO: 2). Incertain embodiments, ANGPTL3 has the sequence as set forth in GenBankAccession No. BG562798.1 (incorporated herein as SEQ ID NO: 3). Incertain embodiments, ANGPTL3 has the sequence as set forth in GenBankAccession No. NM_(—)014495.1 (incorporated herein as SEQ ID NO: 4). Incertain embodiments, ANGPTL3 has the sequence as set forth in GenBankAccession No. NT 032977.5 nucleotides 15511702 to Ser. No. 15/521,082(incorporated herein as SEQ ID NO: 5). In certain embodiments, ANGPTL3has the sequence as set forth in GenBank Accession No. AF162224.1(incorporated herein as SEQ ID NO: 6). In certain embodiments, ANGPTL3has the sequence as set forth in GenBank Accession No. AI195524.1(incorporated herein as SEQ ID NO: 7). In certain embodiments, ANGPTL3has the sequence as set forth in GenBank Accession No. BB717501.1(incorporated herein as SEQ ID NO: 8).

TABLE 1 Gene Target Names and Sequences SEQ Target Name Species Genbank# ID NO angiopoietin-like 3 Human BG400407.1 1 angiopoietin-like 3 HumanBG562555.1 2 angiopoietin-like 3 Human BG562798.1 3 angiopoietin-like 3Human NM_014495.1 4 angiopoietin-like 3 Human nucleotides 15511702 to 515521082 of NT_032977.5 angiopoietin-like 3 Mouse AF162224.1 6angiopoietin-like 3 Mouse AI195524.1 7 angiopoietin-like 3 MouseBB717501.1 8

In certain embodiments, the animal is a human.

In certain embodiments, the compounds or compositions of the inventionare designated as a first agent and the methods or uses of the inventionfurther comprise administering a second agent.

In certain embodiments, the first agent and the second agent areco-administered. In certain embodiments the first agent and the secondagent are co-administered sequentially or concomitantly.

In certain embodiments, the second agent is a glucose-lowering agent.The glucose lowering agent can include, but is not limited to, atherapeutic lifestyle change, PPAR agonist, a dipeptidyl peptidase (IV)inhibitor, a GLP-1 analog, insulin or an insulin analog, an insulinsecretagogue, a SGLT2 inhibitor, a human amylin analog, a biguanide, analpha-glucosidase inhibitor, or a combination thereof. Theglucose-lowering agent can include, but is not limited to metformin,sulfonylurea, rosiglitazone, meglitinide, thiazolidinedione,alpha-glucosidase inhibitor or a combination thereof. The sulfonylureacan be acetohexamide, chlorpropamide, tolbutamide, tolazamide,glimepiride, a glipizide, a glyburide, or a gliclazide. The meglitinidecan be nateglinide or repaglinide. The thiazolidinedione can bepioglitazone or rosiglitazone. The alpha-glucosidase can be acarbose ormiglitol.

In certain embodiments, the second agent is a lipid-lowering therapy. Incertain embodiments the lipid lowering therapy can include, but is notlimited to, a therapeutic lifestyle change, HMG-CoA reductase inhibitor,cholesterol absorption inhibitor, MTP inhibitor, antisense compoundtargeted to ApoB or any combination thereof. The HMG-CoA reductaseinhibitor can be atorvastatin, rosuvastatin, fluvastatin, lovastatin,pravastatin, or simvastatin. The cholesterol absorption inhibitor can beezetimibe.

In certain embodiments, administration comprises parenteraladministration.

In certain embodiments, the metabolic or cardiovascular diseaseincludes, but is not limited to, obesity, diabetes, atherosclerosis,dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease(NAFLD), hyperfattyacidemia or metabolic syndrome, or a combinationthereof. The dyslipidemia can be hyperlipidemia. The hyperlipidemia canbe hypercholesterolemia, hypertriglyceridemia, or bothhypercholesterolemia and hypertriglyceridemia. The NAFLD can be hepaticsteatosis or steatohepatitis. The diabetes can be type 2 diabetes ortype 2 diabetes with dyslipidemia.

In certain embodiments, administering the compound of the inventionresults in a reduction of lipid levels, including triglyceride levels,cholesterol levels, insulin resistance, glucose levels or a combinationthereof. One or more of the levels can be independently reduced by 5%,10%, 20%, 30%, 35%, or 40%. Administering the compound of the inventioncan result in improved insulin sensitivity or hepatic insulinsensitivity. Administering the compound of the invention can result in areduction in atherosclerotic plaques, obesity, glucose, lipids, glucoseresistance, cholesterol, or improvement in insulin sensitivity or anycombination thereof.

Certain embodiments provide the use of a compound as described herein inthe manufacture of a medicament for treating, ameliorating, delaying orpreventing one or more of a metabolic disease or a cardiovasculardisease.

Certain embodiments provide a kit for treating, preventing, orameliorating one or more of a metabolic disease or a cardiovasculardisease as described herein wherein the kit comprises: a) a compound asdescribed herein; and optionally b) an additional agent or therapy asdescribed herein. The kit can further include instructions or a labelfor using the kit to treat, prevent, or ameliorate one or more of ametabolic disease or a cardiovascular disease.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense compounds, antisense oligonucleotides, and siRNAs. Anoligomeric compound can be “antisense” to a target nucleic acid, meaningthat is capable of undergoing hybridization to a target nucleic acidthrough hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an antisense oligonucleotide hasa nucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to ANGPTL3nucleic acid is 10 to 30 nucleotides in length. In other words,antisense compounds are from 10 to 30 linked nucleobases. In otherembodiments, the antisense compound comprises a modified oligonucleotideconsisting of 8 to 80, 10-80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or20 linked nucleobases. In certain such embodiments, the antisensecompound comprises a modified oligonucleotide consisting of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linkednucleobases in length, or a range defined by any two of the abovevalues.

In certain embodiments, the antisense compound comprises a shortened ortruncated modified oligonucleotide. The shortened or truncated modifiedoligonucleotide can have a single nucleoside deleted from the 5′ end (5′truncation), or alternatively from the 3′ end (3′ truncation). Ashortened or truncated oligonucleotide can have two or more nucleosidesdeleted from the 5′ end, or alternatively can have two or morenucleosides deleted from the 3′ end. Alternatively, the deletednucleosides can be dispersed throughout the modified oligonucleotide,for example, in an antisense compound having one or more nucleosidedeleted from the 5′ end and one or more nucleoside deleted from the 3′end.

When a single additional nucleoside is present in a lengthenedoligonucleotide, the additional nucleoside can be located at the 5′, 3′end or central portion of the oligonucleotide. When two or moreadditional nucleosides are present, the added nucleosides can beadjacent to each other, for example, in an oligonucleotide having twonucleosides added to the 5′ end (5′ addition), or alternatively to the3′ end (3′ addition) or the central portion, of the oligonucleotide.Alternatively, the added nucleoside can be dispersed throughout theantisense compound, for example, in an oligonucleotide having one ormore nucleoside added to the 5′ end, one or more nucleoside added to the3′ end, and/or one or more nucleoside added to the central portion.

It is possible to increase or decrease the length of an antisensecompound, such as an antisense oligonucleotide, and/or introducemismatch bases without eliminating activity. For example, in Woolf etal. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series ofantisense oligonucleotides 13-25 nucleobases in length were tested fortheir ability to induce cleavage of a target RNA in an oocyte injectionmodel. Antisense oligonucleotides 25 nucleobases in length with 8 or 11mismatch bases near the ends of the antisense oligonucleotides were ableto direct specific cleavage of the target mRNA, albeit to a lesserextent than the antisense oligonucleotides that contained no mismatches.Similarly, target specific cleavage was achieved using 13 nucleobaseantisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a seriesof tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42nucleobase antisense oligonucleotides comprised of the sequence of twoor three of the tandem antisense oligonucleotides, respectively, fortheir ability to arrest translation of human DHFR in a rabbitreticulocyte assay. Each of the three 14 nucleobase antisenseoligonucleotides alone was able to inhibit translation, albeit at a moremodest level than the 28 or 42 nucleobase antisense oligonucleotides.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to an ANGPTL3nucleic acid have chemically modified subunits arranged in patterns, ormotifs, to confer to the antisense compounds properties such as enhancedinhibitory activity, increased binding affinity for a target nucleicacid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region of achimeric antisense compound can optionally serve as a substrate for thecellular endonuclease RNase H, which cleaves the RNA strand of anRNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimericantisense compounds. In a gapmer an internal region having a pluralityof nucleotides that supports RNaseH cleavage is positioned betweenexternal regions having a plurality of nucleotides that are chemicallydistinct from the nucleosides of the internal region. In the case of anantisense oligonucleotide having a gapmer motif, the gap segmentgenerally serves as the substrate for endonuclease cleavage, while thewing segments comprise modified nucleosides. In certain embodiments, theregions of a gapmer are differentiated by the types of sugar moietiescomprising each distinct region. The types of sugar moieties that areused to differentiate the regions of a gapmer can in some embodimentsinclude β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modifiednucleosides (such 2′-modified nucleosides can include 2′-MOE, and2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (suchbicyclic sugar modified nucleosides can include those having a4′-(CH2)n-O-2′ bridge, where n=1 or n=2). Preferably, each distinctregion comprises uniform sugar moieties. The wing-gap-wing motif isfrequently described as “X—Y—Z”, where “X” represents the length of the5′ wing region, “Y” represents the length of the gap region, and “Z”represents the length of the 3′ wing region. As used herein, a gapmerdescribed as “X—Y—Z” has a configuration such that the gap segment ispositioned immediately adjacent each of the 5′ wing segment and the 3′wing segment. Thus, no intervening nucleotides exist between the 5′ wingsegment and gap segment, or the gap segment and the 3′ wing segment. Anyof the antisense compounds described herein can have a gapmer motif. Insome embodiments, X and Z are the same, in other embodiments they aredifferent. In a preferred embodiment, Y is between 8 and 15 nucleotides.X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morenucleotides. Thus, gapmers include, but are not limited to, for example5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3,2-10-2, 1-10-1, 2-8-2, 6-8-6, 5-8-5, 1-8-1, 2-6-2, 6-8-6, 5-8-5, 1-8-1,2-6-2, 2-13-2, 1-8-2, 2-8-3, 3-10-2, 1-18-2, or 2-18-2.

In certain embodiments, the antisense compound as a “wingmer” motif,having a wing-gap or gap-wing configuration, i.e. an X—Y or Y—Zconfiguration as described above for the gapmer configuration. Thus,wingmer configurations include, but are not limited to, for example5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, or5-13.

In certain embodiments, antisense compounds targeted to an ANGPTL3nucleic acid possess a 5-10-5 gapmer motif.

In certain embodiments, an antisense compound targeted to an ANGPTL3nucleic acid has a gap-widened motif.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode ANGPTL3 include, without limitation,the following: the human sequence as set forth in GenBank Accession No.BG400407.1 (incorporated herein as SEQ ID NO: 1), GenBank Accession No.BG562555.1 (incorporated herein as SEQ ID NO: 2), GenBank Accession No.BG562798.1 (incorporated herein as SEQ ID NO: 3), GenBank Accession No.NM_(—)014495.1 (incorporated herein as SEQ ID NO: 4), GenBank AccessionNo. NT 032977.5 nucleotides 15511702 to Ser. No. 15/521,082(incorporated herein as SEQ ID NO: 5), GenBank Accession No. AF162224.1(incorporated herein as SEQ ID NO: 6), GenBank Accession No. AI195524.1(incorporated herein as SEQ ID NO: 7) and GenBank Accession No.BB717501.1 (incorporated herein as SEQ ID NO: 8). It is understood thatthe sequence set forth in each SEQ ID NO in the Examples containedherein is independent of any modification to a sugar moiety, aninternucleoside linkage, or a nucleobase. As such, antisense compoundsdefined by a SEQ ID NO can comprise, independently, one or moremodifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis No)indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined regionof the target nucleic acid. For example, a target region can encompass a3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region. The structurally defined regionsfor ANGPTL3 can be obtained by accession number from sequence databasessuch as NCBI and such information is incorporated herein by reference.In certain embodiments, a target region can encompass the sequence froma 5′ target site of one target segment within the target region to a 3′target site of another target segment within the target region.

In certain embodiments, a “target segment” is a smaller, sub-portion ofa target region within a nucleic acid. For example, a target segment canbe the sequence of nucleotides of a target nucleic acid to which one ormore antisense compound is targeted. “5′ target site” refers to the5′-most nucleotide of a target segment. “3′ target site” refers to the3′-most nucleotide of a target segment.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In certain embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

A target region can contain one or more target segments. Multiple targetsegments within a target region can be overlapping. Alternatively, theycan be non-overlapping. In certain embodiments, target segments within atarget region are separated by no more than about 300 nucleotides. Incertain embodiments, target segments within a target region areseparated by a number of nucleotides that is, is about, is no more than,is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, or 10 nucleotides on the target nucleic acid, or is a range definedby any two of the preceding values. In certain embodiments, targetsegments within a target region are separated by no more than, or nomore than about, 5 nucleotides on the target nucleic acid. In certainembodiments, target segments are contiguous. Contemplated are targetregions defined by a range having a starting nucleic acid that is any ofthe 5′ target sites or 3′ target sites listed herein.

Suitable target segments can be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, an exon, or an exon/intron junction. Targetsegments containing a start codon or a stop codon are also suitabletarget segments. A suitable target segment can specifically exclude acertain structurally defined region such as the start codon or stopcodon.

The determination of suitable target segments can include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm can be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that canhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There can be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin an active target region. In certain embodiments, reductions inANGPTL3 mRNA levels are indicative of inhibition of ANGPTL3 proteinexpression.

Reductions in levels of an ANGPTL3 protein are also indicative ofinhibition of target mRNA expression. Further, phenotypic changes, suchas a reduction of the level of cholesterol, LDL, triglyceride, orglucose, can be indicative of inhibition of ANGPTL3 mRNA and/or proteinexpression.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and a ANGPTL3 nucleic acid. The most common mechanismof hybridization involves hydrogen bonding (e.g., Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art (Sambrooke andRussell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). Incertain embodiments, the antisense compounds provided herein arespecifically hybridizable with an ANGPTL3 nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as an ANGPTL3nucleic acid).

An antisense compound can hybridize over one or more segments of anANGPTL3 nucleic acid such that intervening or adjacent segments are notinvolved in the hybridization event (e.g., a loop structure, mismatch orhairpin structure).

In certain embodiments, the antisense compounds provided herein, or aspecified portion thereof, are, or are at least, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% complementary to an ANGPTL3 nucleic acid, a target region, targetsegment, or specified portion thereof. In certain embodiments, theantisense compounds provided herein, or a specified portion thereof,are, or are at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to thesequence of one or more of SEQ ID NOs: 1-5. Percent complementarity ofan antisense compound with a target nucleic acid can be determined usingroutine methods.

For example, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases can be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden,Genome Res., 1997, 7, 649 656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, orspecified portions thereof, are fully complementary (i.e. 100%complementary) to a target nucleic acid, or specified portion thereof.For example, an antisense compound can be fully complementary to anANGPTL3 nucleic acid, or a target region, or a target segment or targetsequence thereof. As used herein, “fully complementary” means eachnucleobase of an antisense compound is capable of precise base pairingwith the corresponding nucleobases of a target nucleic acid. Forexample, a 20 nucleobase antisense compound is fully complementary to atarget sequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the antisense compound. Fully complementary canalso be used in reference to a specified portion of the first and/or thesecond nucleic acid. For example, a 20 nucleobase portion of a 30nucleobase antisense compound can be “fully complementary” to a targetsequence that is 400 nucleobases long. The 20 nucleobase portion of the30 nucleobase oligonucleotide is fully complementary to the targetsequence if the target sequence has a corresponding 20 nucleobaseportion wherein each nucleobase is complementary to the 20 nucleobaseportion of the antisense compound. At the same time, the entire 30nucleobase antisense compound can be fully complementary to the targetsequence, depending on whether the remaining 10 nucleobases of theantisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase can be at the 5′ end or3′ end of the antisense compound. Alternatively, the non-complementarynucleobase or nucleobases can be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they can be either contiguous (i.e. linked) or non-contiguous.In one embodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 10,12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas an ANGPTL3 nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 10,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleobases in length comprise no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas an ANGPTL3 nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In certain embodiments, the antisensecompounds, are complementary to at least an 8 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 10 nucleobase portion of a target segment.In certain embodiments, the antisense compounds are complementary to atleast a 15 nucleobase portion of a target segment. Also contemplated areantisense compounds that are complementary to at least an 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of atarget segment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein can also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or the sequenceof a compound represented by a specific Isis number, or portion thereof.As used herein, an antisense compound is identical to the sequencedisclosed herein if it has the same nucleobase pairing ability. Forexample, a RNA which contains uracil in place of thymidine in adisclosed DNA sequence would be considered identical to the DNA sequencesince both uracil and thymidine pair with adenine. Shortened andlengthened versions of the antisense compounds described herein as wellas compounds having non-identical bases relative to the antisensecompounds provided herein also are contemplated. The non-identical basescan be adjacent to each other or dispersed throughout the antisensecompound. Percent identity of an antisense compound is calculatedaccording to the number of bases that have identical base pairingrelative to the sequence to which it is being compared.

In certain embodiments, the antisense compounds, or portions thereof,are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to one or more of the antisense compounds or SEQ ID NOs, or aportion thereof, disclosed herein.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides can also be employed to increase thebinding affinity of a shortened or truncated antisense oligonucleotidefor its target nucleic acid. Consequently, comparable results can oftenbe obtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, antisense compounds targeted to an ANGPTL3nucleic acid comprise one or more modified internucleoside linkages. Incertain embodiments, the modified internucleoside linkages arephosphorothioate linkages. In certain embodiments, each internucleosidelinkage of an antisense compound is a phosphorothioate internucleosidelinkage.

Modified Sugar Moieties

Antisense compounds can optionally contain one or more nucleosideswherein the sugar group has been modified. Such sugar modifiednucleosides can impart enhanced nuclease stability, increased bindingaffinity or some other beneficial biological property to the antisensecompounds. In certain embodiments, nucleosides comprise a chemicallymodified ribofuranose ring moieties. Examples of chemically modifiedribofuranose rings include without limitation, addition of substitutentgroups (including 5′ and 2′ substituent groups, bridging of non-geminalring atoms to form bicyclic nucleic acids (BNA), replacement of theribosyl ring oxygen atom with S, N(R), or C(R1)(R)2 (R=H, C1-C12 alkylor a protecting group) and combinations thereof. Examples of chemicallymodified sugars include 2′-F-5′-methyl substituted nucleoside (see PCTInternational Application WO 2008/101157 Published on Aug. 21, 2008 forother disclosed 5′,2′-bis substituted nucleosides) or replacement of theribosyl ring oxygen atom with S with further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA(see PCT International Application WO 2007/134181 Published on Nov. 22,2007 wherein LNA is substituted with for example a 5′-methyl or a5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH3 and 2′-O(CH2)2OCH3 substituent groups. The substituent atthe 2′ position can also be selected from allyl, amino, azido, thio,0-allyl, 0-C1-C10 alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-O—N(Rm)(Rn), andO—CH2-C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H orsubstituted or unsubstituted C1-C10 alkyl.

Examples of bicyclic nucleic acids (BNAs) include without limitationnucleosides comprising a bridge between the 4′ and the 2′ ribosyl ringatoms. In certain embodiments, antisense compounds provided hereininclude one or more BNA nucleosides wherein the bridge comprises one ofthe formulas: 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2; 4′-(CH2)2-O-2′ (ENA);4′-C(CH3)2-O-2′ (see PCT/US2008/068922); 4′-CH(CH3)-O-2′ and4′-C—H(CH2OCH3)

-O-2′ (see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008);4′-CH2-N(OCH3)-2′ (see PCT/US2008/064591); 4′-CH2-O—N(CH3)-2′ (seepublished U.S. Patent Application US2004-0171570, published Sep. 2,2004); 4′-CH2-N(R)—O-2′ (see U.S. Pat. No. 7,427,672, issued on Sep. 23,2008); 4′-CH2-CH (see Chattopadhyaya et al., J. Org. Chem, 2009, 74,118-134) (CH3)-2′ and 4′-CH2-C

(═CH2)-2′ (see PCT/US2008/066154); and wherein R is, independently, H,C1-C12 alkyl, or a protecting group. Each of the foregoing BNAs includevarious stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see PCT international applicationPCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226). Previously,α-L-methyleneoxy (4′-CH₂—O-2′) BNA's have also been incorporated intoantisense oligonucleotides that showed antisense activity (Frieden etal., Nucleic Acids Research, 2003, 21, 6365-6372).

Further reports related to bicyclic nucleosides can be found inpublished literature (see for example: Srivastava et al., J. Am. Chem.Soc., 2007, 129, 8362-8379; U.S. Pat. Nos. 7,053,207; 6,268,490;6,770,748; 6,794,499; 7,034,133; and 6,525,191; Elayadi et al., Curr.Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol.,2001, 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3,239-243; and U.S. Pat. No. 6,670,461; International applications WO2004/106356; WO 94/14226; WO 2005/021570; U.S. Patent Publication Nos.US2004-0171570; US2007-0287831; US2008-0039618; U.S. Pat. No. 7,399,845;U.S. patent Ser. Nos. 12/129,154; 60/989,574; 61/026,995; 61/026,998;61/056,564; 61/086,231; 61/097,787; 61/099,844; PCT InternationalApplications Nos. PCT/US2008/064591; PCT/US2008/066154;PCT/US2008/068922; and Published PCT International Applications WO2007/134181).

In certain embodiments, bicyclic sugar moieties of BNA nucleosidesinclude, but are not limited to, compounds having at least one bridgebetween the 4′ and the 2′ position of the pentofuranosyl sugar moietywherein such bridges independently comprises 1 or from 2 to 4 linkedgroups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═O)—, —C(═NR_(a))—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl ora protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is,—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certainembodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′,4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′-wherein each R is, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH₂—N(R)—O-2) BNA, and (F)Methyl(methyleneoxy) (4′-CH(CH₃)—O-2) BNA, (G) Methylene-thio(4′-CH₂—S-2′) BNA, (H) Methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) Methylcarbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, and (J) Propylene carbocyclic(4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is the base moiety and R is independently H, a protectinggroup or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleoside having Formula I:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium.

In certain embodiments, bicyclic nucleoside having Formula II:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl,substituted acyl, substituted amide, thiol or substituted thio.

In one embodiment, each of the substituted groups is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃,OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) andJ_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl andX is O or NJ_(c).

In certain embodiments, bicyclic nucleoside having Formula III:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl orsubstituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleoside having Formula IV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl,substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl orsubstituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleoside having Formula V:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen,halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j),SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k),C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

-   -   q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂        alkyl or substituted C₁-C₁₂ alkyl.

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNAmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (Koshkin et al., Tetrahedron, 1998, 54,3607-3630). BNAs and preparation thereof are also described in WO98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA and 2′-thio-BNAs, have alsobeen prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs comprisingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., WO 99/14226).Furthermore, synthesis of 2′-amino-BNA, a novel comformationallyrestricted high-affinity oligonucleotide analog has been described inthe art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Inaddition, 2′-amino- and 2′-methylamino-BNA's have been prepared and thethermal stability of their duplexes with complementary RNA and DNAstrands has been previously reported.

In certain embodiments, bicyclic nucleoside having Formula VI:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) orN(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Freier etal., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al.,J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation ofcarbocyclic bicyclic nucleosides along with their oligomerization andbiochemical studies have also been described (Srivastava et al., J. Am.Chem. Soc., 2007, 129(26), 8362-8379).

In certain embodiments, nucleosides are modified by replacement of theribosyl ring with a sugar surrogate. Such modification includes withoutlimitation, replacement of the ribosyl ring with a surrogate ring system(sometimes referred to as DNA analogs) such as a morpholino ring, acyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring such asone having one of the formula:

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknow in the art that can be used to modify nucleosides for incorporationinto antisense compounds (see for example review article: Leumann,Christian J., Bioorganic & Medicinal Chemistry, 2002, 10, 841-854). Suchring systems can undergo various additional substitutions to enhanceactivity. See for example compounds having Formula VII:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VII:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, an internucleoside linkinggroup linking the tetrahydropyran nucleoside analog to the antisensecompound or one of T_(a) and T_(b) is an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundand the other of T_(a) and T_(b) is H, a hydroxyl protecting group, alinked conjugate group or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl; and each of R₁ and R₂ is selectedfrom hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein Xis O, S or NJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H (M). Incertain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ isother than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄,q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides ofFormula VII are provided wherein one of R₁ and R₂ is fluoro (K). Incertain embodiments, THP nucleosides of Formula VII are provided whereinone of R₁ and R₂ is methoxyethoxy. In certain embodiments, R₁ is fluoroand R₂ is H; R₁ is H and R₂ is fluoro; R₁ is methoxy and R₂ is H, and R₁is H and R₂ is methoxyethoxy. Methods for the preparations of modifiedsugars are well known to those skilled in the art.

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds targeted to an ANGPTL3nucleic acid comprise one or more nucleotides having modified sugarmoieties. In certain embodiments, the modified sugar moiety is 2′-MOE.In certain embodiments, the 2′-MOE modified nucleotides are arranged ina gapmer motif. In certain embodiments, the modified sugar moiety is abicyclic nucleoside having a (4′-CH(CH₃)—O-2′) bridging group. Incertain embodiments, the (4′-CH(CH₃)—O-2′) modified nucleotides arearranged throughout the wings of a gapmer motif.

Methods for the preparations of modified sugars are well known to thoseskilled in the art.

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds targeted to an ANGPTL3nucleic acid comprise one or more nucleotides having modified sugarmoieties. In certain embodiments, the modified sugar moiety is 2′-MOE.In certain embodiments, the 2′-MOE modified nucleotides are arranged ina gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified nucleobases. Both natural and modifiednucleobases are capable of participating in hydrogen bonding. Suchnucleobase modifications can impart nuclease stability, binding affinityor some other beneficial biological property to antisense compounds.Modified nucleobases include synthetic and natural nucleobases such as,for example, 5-methylcytosine (5-me-C). Certain nucleobasesubstitutions, including 5-methylcytosine substitutions, areparticularly useful for increasing the binding affinity of an antisensecompound for a target nucleic acid. For example, 5-methylcytosinesubstitutions have been shown to increase nucleic acid duplex stabilityby 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds.,Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties can also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases that are particularly useful for increasing the bindingaffinity of antisense compounds include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to an ANGPTL3nucleic acid comprise one or more modified nucleobases. In certainembodiments, shortened or gap-widened antisense oligonucleotidestargeted to an ANGPTL3 nucleic acid comprise one or more modifiednucleobases. In certain embodiments, the modified nucleobase is5-methylcytosine. In certain embodiments, each cytosine is a5-methylcytosine.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides can be admixed with pharmaceuticallyacceptable active or inert substance for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Antisense compound targeted to an ANGPTL3 nucleic acid can be utilizedin pharmaceutical compositions by combining the antisense compound witha suitable pharmaceutically acceptable diluent or carrier. Apharmaceutically acceptable diluent includes phosphate-buffered saline(PBS). PBS is a diluent suitable for use in compositions to be deliveredparenterally. Accordingly, in one embodiment, employed in the methodsdescribed herein is a pharmaceutical composition comprising an antisensecompound targeted to an ANGPTL3 nucleic acid and a pharmaceuticallyacceptable diluent. In certain embodiments, the pharmaceuticallyacceptable diluent is PBS. In certain embodiments, the antisensecompound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other oligonucleotide which, upon administration to an animal,including a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to pharmaceutically acceptablesalts of antisense compounds, prodrugs, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents. Suitablepharmaceutically acceptable salts include, but are not limited to,sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an antisense compound which are cleaved by endogenousnucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds can be covalently linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the resulting antisense oligonucleotides. Typical conjugategroups include cholesterol moieties and lipid moieties. Additionalconjugate groups include carbohydrates, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizinggroups that are generally attached to one or both termini of antisensecompounds to enhance properties such as, for example, nucleasestability. Included in stabilizing groups are cap structures. Theseterminal modifications protect the antisense compound having terminalnucleic acids from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be presenton both termini. Cap structures are well known in the art and include,for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizinggroups that can be used to cap one or both ends of an antisense compoundto impart nuclease stability include those disclosed in WO 03/004602published on Jan. 16, 2003.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expressionof ANGPTL3 nucleic acids can be tested in vitro in a variety of celltypes. Cell types used for such analyses are available from commercialvendors (e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio,Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville,Md.) and cells are cultured according to the vendor's instructions usingcommercially available reagents (e.g. Invitrogen Life Technologies,Carlsbad, Calif.). Illustrative cell types include, but are not limitedto, HepG2 cells, Hep3B cells, Huh7 (hepatocellular carcinoma) cells,primary hepatocytes, A549 cells, GM04281 fibroblasts and LLC-MK2 cells.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisenseoligonucleotides, which can be modified appropriately for treatment withother antisense compounds.

In general, cells are treated with antisense oligonucleotides when thecells reach approximately 60-80% confluence in culture.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotidesare mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.)to achieve the desired final concentration of antisense oligonucleotideand a LIPOFECTIN® concentration that typically ranges 2 to 12 ug/mL per100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE 2000® (Invitrogen, Carlsbad,Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE 2000® inOPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) toachieve the desired concentration of antisense oligonucleotide and aLIPOFECTAMINE® concentration that typically ranges 2 to 12 ug/mL per 100nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes Cytofectin® (Invitrogen, Carlsbad, Calif.).Antisense oligonucleotide is mixed with Cytofectin® in OPTI-MEM® 1reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve thedesired concentration of antisense oligonucleotide and a Cytofectin®concentration that typically ranges 2 to 12 ug/mL per 100 nM antisenseoligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes Oligofectamine™ (Invitrogen Life Technologies,Carlsbad, Calif.). Antisense oligonucleotide is mixed withOligofectamine™ in Opti-MEM™-1 reduced serum medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to achieve the desired concentration ofoligonucleotide with an Oligofectamine™ to oligonucleotide ratio ofapproximately 0.2 to 0.8 μL per 100 nM.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes FuGENE 6 (Roche Diagnostics Corp., Indianapolis,Ind.). Antisense oligomeric compound was mixed with FuGENE 6 in 1 mL ofserum-free RPMI to achieve the desired concentration of oligonucleotidewith a FuGENE 6 to oligomeric compound ratio of 1 to 4 μL of FuGENE 6per 100 nM.

Another technique used to introduce antisense oligonucleotides intocultured cells includes electroporation (Sambrooke and Russell,Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001).

Cells are treated with antisense oligonucleotides by routine methods.Cells are typically harvested 16-24 hours after antisenseoligonucleotide treatment, at which time RNA or protein levels of targetnucleic acids are measured by methods known in the art and describedherein. In general, when treatments are performed in multiplereplicates, the data are presented as the average of the replicatetreatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line.

Methods to determine the optimal antisense oligonucleotide concentrationfor a particular cell line are well known in the art. Antisenseoligonucleotides are typically used at concentrations ranging from 1 nMto 300 nM when transfected with LIPOFECTAMINE2000®, Lipofectin orCytofectin. Antisense oligonucleotides are used at higher concentrationsranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+mRNA.Methods of RNA isolation are well known in the art (Sambrooke andRussell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). RNAis prepared using methods well known in the art, for example, using theTRIZOL® Reagent (Invitrogen, Carlsbad, Calif.) according to themanufacturer's recommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of an ANGPTL3 nucleic acid can beassayed in a variety of ways known in the art (Sambrooke and Russell,Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). For example,target nucleic acid levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), or quantitativereal-time PCR. RNA analysis can be performed on total cellular RNA orpoly(A)+mRNA. Methods of RNA isolation are well known in the art.Northern blot analysis is also routine in the art. Quantitativereal-time PCR can be conveniently accomplished using the commerciallyavailable ABI PRISM® 7600, 7700, or 7900 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels can be accomplished by quantitativereal-time PCR using the ABI PRISM® 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. Methods of quantitative real-time PCRare well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT, and real-time-PCR reactions arecarried out by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR can benormalized using either the expression level of a gene whose expressionis constant, such as cyclophilin A, or by quantifying total RNA usingRIBOGREEN® (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expressionis quantified by real time PCR, by being run simultaneously with thetarget, multiplexing, or separately. Total RNA is quantified usingRIBOGREEN® RNA quantification reagent (Invitrogen, Inc. Carlsbad,Calif.). Methods of RNA quantification by RIBOGREEN® are taught inJones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). ACYTOFLUOR® 4000 instrument (PE Applied Biosystems) is used to measureRIBOGREEN® fluorescence.

Probes and primers are designed to hybridize to an ANGPTL3 nucleic acid.Methods for designing real-time PCR probes and primers are well known inthe art, and can include the use of software such as PRIMER EXPRESS®Software (Applied Biosystems, Foster City, Calif.).

Gene target quantities obtained by RT, real-time PCR can be normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression was quantified by RT,real-time PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA was quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).

Presented in Table 2 are primers and probes used to measure GAPDHexpression in the cell types described herein. The GAPDH PCR probes haveJOE covalently linked to the 5′ end and TAMRA or MGB covalently linkedto the 3′ end, where JOE is the fluorescent reporter dye and TAMRA orMGB is the quencher dye. In some cell types, primers and probe designedto a GAPDH sequence from a different species are used to measure GAPDHexpression. For example, a human GAPDH primer and probe set is used tomeasure GAPDH expression in monkey-derived cells and cell lines.

TABLE 2 GAPDH primers and probes for use in real-time PCR SEQ TargetSequence ID Name Species Description Sequence (5′ to 3′) NO GAPDH HumanForward Primer CAACGGATTTGGTCGTATTGG 15 GAPDH Human Reverse PrimerGGCAACAATATCCACTTTACCAGAGT 16 GAPDH Human Probe CGCCTGGTCACCAGGGCTGCT 17GAPDH Human Forward Primer GAAGGTGAAGGTCGGAGTC 18 GAPDH HumanReverse Primer GAAGATGGTGATGGGATTTC 19 GAPDH Human ProbeCAAGCTTCCCGTTCTCAGCC 20 GAPDH Human Forward Primer GAAGGTGAAGGTCGGAGTC18 GAPDH Human Reverse Primer GAAGATGGTGATGGGATTTC 19 GAPDH Human ProbeTGGAATCATATTGGAACATG 21 GAPDH Mouse Forward Primer GGCAAATTCAACGGCACAGT22 GAPDH Mouse Reverse Primer GGGTCTCGCTCCTGGAAGAT 23 GAPDH Mouse ProbeAAGGCCGAGAATGGGAAGCTTGTCATC 24 GAPDH Rat Forward PrimerTGTTCTAGAGACAGCCGCATCTT 25 GAPDH Rat Reverse PrimerCACCGACCTTCACCATCTTGT 26 GAPDH Rat Probe TTGTGCAGTGCCAGCCTCGTCTCA 27

Probes and primers for use in real-time PCR were designed to hybridizeto target-specific sequences. The primers and probes and the targetnucleic acid sequences to which they hybridize are presented in Table 3.The target-specific PCR probes have FAM covalently linked to the 5′ endand TAMRA or MGB covalently linked to the 3′ end, where FAM is thefluorescent dye and TAMRA or MGB is the quencher dye.

TABLE 3 Gene target-specific primers and probes for use in real-time PCRTarget SEQ SEQ Sequence ID Species ID NO Description Sequence (5′ to 3′)NO Human 4 Forward Primer CTTCAATGAAACGTGGGAGAACT 28 Human 4Reverse Primer TCTCTAGGCCCAACCAAAATTC 29 Human 4 ProbeAAATATGGTTTTGGGAGGCTTGAT 30 Mouse 6 Forward PrimerCAGAAGTAACATCACTCAAAAGTTTTGTAG 31 Mouse 6 Reverse PrimerGACTTAATTGTTTATACTGTTCTTCCACACT 32 Mouse 6 ProbeCAGCAAGACAACAGCATAAGAGAACTCCTCCA 33

Analysis of Protein Levels

Antisense inhibition of ANGPTL3 nucleic acids can be assessed bymeasuring ANGPTL3 protein levels. Protein levels of ANGPTL3 can beevaluated or quantitated in a variety of ways well known in the art,such as immunoprecipitation, Western blot analysis (immunoblotting),enzyme-linked immunosorbent assay (ELISA), quantitative protein assays,protein activity assays (for example, caspase activity assays),immunohistochemistry, immunocytochemistry or fluorescence-activated cellsorting (FACS) (Sambrooke and Russell, Molecular Cloning: A LaboratoryManual, 3^(rd) Ed., 2001). Antibodies directed to a target can beidentified and obtained from a variety of sources, such as the MSRScatalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can beprepared via conventional monoclonal or polyclonal antibody generationmethods well known in the art.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are testedin animals to assess their ability to inhibit expression of ANGPTL3 andproduce phenotypic changes. Testing can be performed in normal animals,or in experimental disease models. For administration to animals,antisense oligonucleotides are formulated in a pharmaceuticallyacceptable diluent, such as phosphate-buffered saline. Administrationincludes parenteral routes of administration. Following a period oftreatment with antisense oligonucleotides, RNA is isolated from tissueand changes in ANGPTL3 nucleic acid expression are measured. Changes inANGPTL3 protein levels are also measured.

Certain Indications

In certain embodiments, provided herein are methods of treating anindividual comprising administering one or more pharmaceuticalcompositions as described herein. In certain embodiments, the individualhas a metabolic disease and/or cardiovascular disease. In certainembodiments, the individual has atherosclerosis, hepatic steatosis orhyperlipidemia.

Accordingly, provided herein are methods for ameliorating a symptomassociated with a metabolic disease or cardiovascular disease. Alsoprovided herein are methods for ameliorating a symptom associated withatherosclerosis, hepatic steatosis or hyperlipidemia in a subject inneed thereof. In certain embodiments, provided is a method for reducingthe rate of onset of a symptom associated with a metabolic disease orcardiovascular disease. In certain embodiments, provided is a method forreducing the rate of onset of a symptom associated atherosclerosis,hepatic steatosis or hyperlipidemia. In certain embodiments, provided isa method for reducing the severity of a symptom associated with ametabolic disease or cardiovascular disease. In certain embodiments,provided is a method for reducing the severity of a symptom associatedwith atherosclerosis, hepatic steatosis or hyperlipidemia. In suchembodiments, the methods comprise administering to an individual in needthereof a therapeutically effective amount of a compound targeted to anANGPTL3 nucleic acid.

In certain embodiments, administration of an antisense compound targetedto an ANGPTL3 nucleic acid results in reduction of ANGPTL3 expression byat least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 99%, or a range defined by any two of these values.

In certain embodiments, pharmaceutical compositions comprising anantisense compound targeted to ANGPTL3 are used for the preparation of amedicament for treating a patient suffering from, or susceptible to, ametabolic disease or cardiovascular disease. In certain embodiments,pharmaceutical compositions comprising an antisense compound targeted toANGPTL3 are used for the preparation of a medicament for treating apatient suffering from, or susceptible to, atherosclerosis, hepaticsteatosis or hyperlipidemia.

In certain embodiments, the methods described herein includeadministering a compound comprising a modified oligonucleotide having an8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobaseportion as described herein of a sequence recited in SEQ ID NO: 34-182.

Administration

In certain embodiments, the compounds and compositions as describedherein are administered parenterally.

In certain embodiments, parenteral administration is by infusion.Infusion can be chronic or continuous or short or intermittent. Incertain embodiments, infused pharmaceutical agents are delivered with apump.

In certain embodiments, parenteral administration is by injection. Theinjection can be delivered with a syringe or a pump. In certainembodiments, the injection is a bolus injection. In certain embodiments,the injection is administered directly to a tissue or organ.

Certain Combination Therapies

In certain embodiments, a first agent comprising the modifiedoligonucleotide of the invention is co-administered with one or moresecondary agents. In certain embodiments, such second agents aredesigned to treat the same disease, disorder or condition as the firstagent described herein. In certain embodiments, such second agents aredesigned to treat a different disease, disorder, or condition as thefirst agent described herein. In certain embodiments, such second agentsare designed to treat an undesired side effect of one or morepharmaceutical compositions as described herein. In certain embodiments,second agents are co-administered with the first agent to treat anundesired effect of the first agent. In certain embodiments, secondagents are co-administered with the first agent to produce acombinational effect. In certain embodiments, second agents areco-administered with the first agent to produce a synergistic effect.

In certain embodiments, a first agent and one or more second agents areadministered at the same time. In certain embodiments, the first agentand one or more second agents are administered at different times. Incertain embodiments, the first agent and one or more second agents areprepared together in a single pharmaceutical formulation. In certainembodiments, the first agent and one or more second agents are preparedseparately.

In certain embodiments, second agents include, but are not limited toascorbic acid.

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1 Antisense Inhibition of Human Angiopoietin-Like 3 byOligomeric Compounds

A series of oligomeric compounds was designed to target differentregions of human angiopoietin-like 3, using published sequences cited inTable 1. The compounds are shown in Table 4. All compounds in Table 4are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length,composed of a central “gap” region consisting of 10 2′-deoxynucleotides,which is flanked on both sides (5′ and 3′) by five-nucleotide “wings”.The wings are composed of 2′-O-(2-methoxyethyl) nucleotides, also knownas 2′-MOE nucleotides. The internucleoside (backbone) linkages arephosphorothioate throughout the oligonucleotide. All cytosine residuesare 5-methylcytosines. The oligomeric compounds in Table 4 specificallyhybridize to a target nucleic acid molecule encoding angiopoietin-like 3and are comprised of regions that increase binding affinity, theseregions being the “wings” of the oligomeric compounds. The oligomericcompounds each comprise a region that elicits RNase H activity, thisregions being the “gap” region.

The compounds were analyzed for their effect on gene target mRNA levelsby quantitative real-time PCR as described in other examples herein,using the target-specific primers and probe shown in Table 3 (SEQ ID NO:28, SEQ ID NO: 29, and SEQ ID NO: 30). Data are averages fromexperiments in which Huh7 cells were treated with 150 nM of thedisclosed oligomeric compounds using OLIGOFECTAMINE™. Shown in Table 4is the SEQ ID NO of the sequence to which each oligomeric compound istargeted.

A reduction in expression is expressed as percent inhibition in Table 4.If present, “N.D.” indicates “not determined”. The target regions towhich these oligomeric compounds are inhibitory are herein referred toas “validated target segments.”

TABLE 4 Inhibition of human angiopoietin-like 3 mRNA levels bychimeric oligonucleotides having 2′-MOE wings and deoxy gap TargetSEQ ID Target SEQ ID ISIS # NO Site Sequence (5′ to 3′) % Inhib NO337529 1 548 ATCTGTTGTGATGTCGATAA 66 34 337527 2 336GTATTTAGTCAAGTTTAGAG 39 35 337528 2 429 TATTACAGATTTTTACACAT 21 36337526 3 33 CGTGGAACTGTTTTCTTCTG 63 37 337459 4 22 AGCTTAATTGTGAACATTTT73 38 337460 4 61 ATTCTGGAGGAAATAACTAG 34 39 233675 4 116AAATCTTGATTTTGGCTCTG 61 40 233676 4 121 ATAGCAAATCTTGATTTTGG 52 41337461 4 126 CTAACATAGCAAATCTTGAT 53 42 337462 4 131ATCGTCTAACATAGCAAATC 43 43 337463 4 154 AGGCCATTGGCTAAAATTTT 37 44337464 4 171 CATGTCCCAACTGAAGGAGG 41 45 337465 4 180CTTTAAGACCATGTCCCAAC 0 46 337466 4 203 GCCCTTCGTCTTATGGACAA 28 47 3374674 214 TCATTAATTTGGCCCTTCGT 38 48 337468 4 223 TGAAATATGTCATTAATTTG 0 49233690 4 247 GACTGATCAAATATGTTGAG 53 50 337469 4 271GTTTGCAGCGATAGATCATA 56 51 337470 4 277 TCACTGGTTTGCAGCGATAG 62 52337471 4 364 AGTTCAAGTGACATATTCTT 52 53 337472 4 496AGTGAAGTTACTTCTGGGTG 59 54 337473 4 502 GTTTTAAGTGAAGTTACTTC 50 55337474 4 510 CTACAAAAGTTTTAAGTGAA 25 56 337475 4 558GGTCTTCCACGGTCTGGAGA 70 57 337476 4 624 TAGTCCTTCTGAGCTGATTT 66 58337477 4 637 GGTTCTTGAATACTAGTCCT 80 59 337478 4 648AAATTTCTGTGGGTTCTTGA 74 60 337479 4 665 TGGCTTGGAAGATAGAGAAA 77 61233710 4 683 AGTAGTTCTTGGTGCTCTTG 82 62 337480 4 694TGAAGAAAGGGAGTAGTTCT 60 63 337481 4 701 ATTCAACTGAAGAAAGGGAG 59 64337482 4 710 TCTTATTTCATTCAACTGAA 32 65 337483 4 734AGGAATGCCATCATGTTTTA 39 66 337484 4 762 CTCTGTTATAAATGGTGGTA 65 67337485 4 767 TTCACCTCTGTTATAAATGG 31 68 337486 4 772GTATGTTCACCTCTGTTATA 47 69 337487 4 777 CACTTGTATGTTCACCTCTG 75 70337488 4 782 CATGCCACTTGTATGTTCAC 44 71 337489 4 806AGAGTTGCTGGGTCTGATGG 52 72 337490 4 840 CTGATATAACATCACAGTAG 54 73337491 4 850 CATGGACTACCTGATATAAC 60 74 233717 4 862TGAATTAATGTCCATGGACT 85 75 337492 4 874 TCTATTCGATGTTGAATTAA 13 76337493 4 909 AGTTCTCCCACGTTTCATTG 80 77 337494 4 918CATATTTGTAGTTCTCCCAC 62 78 337495 4 923 AAAACCATATTTGTAGTTCT 25 79337496 4 930 GCCTCCCAAAACCATATTTG 35 80 337497 4 953GCCCAACCAAAATTCTCCAT 70 81 233721 4 959 CTCTAGGCCCAACCAAAATT 73 82233722 4 964 ATCTTCTCTAGGCCCAACCA 91 83 337498 4 995AACATAATTAGATTGCTTCA 26 84 337499 4 1016 GTCTTCCAACTCAATTCGTA 56 85337500 4 1023 CTTTCCAGTCTTCCAACTCA 38 86 337501 4 1094AACTAGATGTAGCGTATAGT 65 87 337502 4 1162 TGATCCCAAGTAGAAAACAC 33 88337503 4 1213 CACCAGCCTCCTGAATAACC 32 89 337504 4 1245TTAGGTTGTTTTCTCCACAC 68 90 337505 4 1301 TAATCCTCTTCTCCTCTCTG 32 91337506 4 1315 TGAGACTTCCAAGATAATCC 63 92 337507 4 1320CATTTTGAGACTTCCAAGAT 48 93 337508 4 1333 GAGTATAACCTTCCATTTTG 54 94337509 4 1364 TGGATGGATCAACATTTTGG 67 95 337510 4 1385TTCAAAGCTTTCTGAATCTG 62 96 337511 4 1397 TGCCTCAGTTCATTCAAAGC 58 97337512 4 1410 TGCCTTTTAAATTTGCCTCA 64 98 337513 4 1443ATTAACTTGGAATGAGGTTA 65 99 337514 4 1450 AGACCACATTAACTTGGAAT 62 100337515 4 1458 AGATTATTAGACCACATTAA 44 101 337516 4 1463ATACCAGATTATTAGACCAC 65 102 337517 4 1470 GGATTTAATACCAGATTATT 64 103337518 5 1678 ACTGACTTACCTGATTTTCT 0 104 337519 5 2294ACCTTGTAAGTCTTCATTGG 52 105 337520 5 3809 CAGTGTTATTCAGATTGTAC 56 106337521 5 4068 AGTGTCTTACCATCATGTTT 56 107 337522 5 5100ACAGATGTAAATAACACTTT 19 108 337523 5 5252 GTCCCCTTACCATCAAGCCT 49 109337524 5 7150 GGGAAGATACTTTGAAGATA 50 110 337525 5 7504CACCAGCCTCCTAAAGGAGA 27 111

Example 2 Antisense Inhibition of Mouse Angiopoietin-Like 3 byOligomeric Compounds

A series of oligomeric compounds was designed to target differentregions of mouse angiopoietin-like 3, using published sequences cited inTable 1. The compounds are shown in Table 5. All compounds in Table 5are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length,composed of a central “gap” region consisting of 10 2′-deoxynucleotides,which is flanked on both sides (5′ and 3′) by five-nucleotide “wings”.The wings are composed of 2′-O-(2-methoxyethyl) nucleotides, also knownas 2′-MOE nucleotides. The internucleoside (backbone) linkages arephosphorothioate throughout the oligonucleotide. All cytosine residuesare 5-methylcytosines. The oligomeric compounds in Table 5 specificallyhybridize to a target nucleic acid molecule encoding angiopoietin-like 3and are comprised of regions that increase binding affinity, theseregions being the “wings” of the oligomeric compounds. The oligomericcompounds each comprise a region that elicits RNase H activity, thisregion being the “gap” region.

The compounds were analyzed for their effect on gene target mRNA levelsby quantitative real-time PCR as described in other examples herein,using the target-specific primers and probe shown in Table 3 (SEQ ID NO:31, SEQ ID NO: 32, and SEQ ID NO: 33). Data are averages fromexperiments in which mouse primary hepatocytes were treated with 150 nMof the disclosed oligomeric compounds using LIPOFECTIN™. The controloligomeric compounds used were SEQ ID NOs: 9 and 10. Shown in Table 5 isthe SEQ ID NO of the sequence to which each oligomeric compound istargeted.

A reduction in expression is expressed as percent inhibition in Table 5.If present, “N.D.” indicates “not determined”. The target regions towhich these oligomeric compounds are inhibitory are herein referred toas “validated target segments”. The antisense oligonucleotides of Table5 may also be cross reactive with the human ANGPTL3 mRNA (GENBANKAccession NM_(—)014495.1, incorporated herein as SEQ ID NO: 4),depending on the number of mismatched nucleobases the murineoligonucleotide has with the human ANGPTL3 sequence. “Human Target StartSite” indicates the 5′-most nucleotide in the human mRNA to which theantisense oligonucleotide is targeted. “Human Target Stop Site”indicates the 3′-most nucleotide in the human mRNA to which theantisense oligonucleotide is targeted. ‘Mismatches’ indicates the numberof nucleobases by which the murine oligonucleotide is mismatched withthe human gene sequence. The designation “n/a” indicates that there wasgreater than 3 mismatches between the murine oligonucleotide and thehuman gene sequence. The greater the complementarity between the murineoligonucleotide and the human gene sequence, the more likely the murineoligonucleotide can cross-react with the human gene sequence.

TABLE 5 Inhibition of mouse angiopoietin-like 3 mRNA levels by chimeric oligonucleotides having 2′-MOE wings and deoxy gap HumanMismatches Target SEQ Target with SEQ Target % ID Start human ISIS #ID NO Site Sequence (5' to 3') Inhib NO Site target 233671 6 19AATAATTTAATTGTGTGCAT 13 112 n/a n/a 233672 6 56 CACTCTGGATGCAATTACTA 66113 n/a n/a 233673 6 68 AAGGTCTGGATCCACTCTGG 74 114 n/a n/a 233674 6 100TTTGGCTCTGAAGGTGCAGA 66 115 n/a n/a 233675 6 110 AAATCTTGATTTTGGCTCTG 8040  116 0 233676 6 115 ATAGCAAATCTTGATTTTGG 49 41  121 0 233677 6 124TCATCCAACATAGCAAATCT 24 116  130 2 233678 6 129 TGACATCATCCAACATAGCA 66117  135 3 233679 6 134 AATTTTGACATCATCCAACA 76 118  140 3 233680 6 139GCTAAAATTTTGACATCATC 72 119  145 2 233681 6 148 AGGCCATTCGCTAAAATTTT 52120  154 1 233682 6 160 CCCAGCTGCAGGAGGCCATT 77 121  166 2 233683 6 165CATGACCCAGCTGCAGGAGG 51 122  171 3 233684 6 172 TTAAGTCCATGACCCAGCTG 71123  178 3 233685 6 182 GACAAAATCTTTAAGTCCAT 27 124  188 2 233686 6 187TTATGGACAAAATCTTTAAG 29 125  193 1 233687 6 226 TTGAGCTTCTGAAATATGTC 56126  232 2 233688 6 231 ATATGTTGAGCTTCTGAAAT 53 127  237 2 233689 6 236ATCAAATATGTTGAGCTTCT 47 128  242 2 233690 6 241 GACTGATCAAATATGTTGAG 7350  247 0 233691 6 266 GGTTCGAAGTGATAGGTCAT 63 129 n/a n/a 233692 6 317TAGTGTAGATGTAGTTCTTC 44 130  323 2 233693 6 349 GACATGTTCTTCACCTCCTC 80131  355 3 233694 6 365 TGAGTTCAGTTCTACTGACA 78 132  371 3 233695 6 373TCAAGCTTTGAGTTCAGTTC 71 133  379 2 233696 6 394 GTCTTCTCTTCCAGCAGACT 71134 n/a n/a 233697 6 405 GTTGAAGGGCTGTCTTCTCT 75 135 n/a n/a 233698 6415 CTGACCTTGTGTTGAAGGGC 92 136 n/a n/a 233699 6 423CCAAAGCCCTGACCTTGTGT 60 137 n/a n/a 233700 6 435 TTAGCTGCTCCTCCAAAGCC 68138 n/a n/a 233701 6 451 CTTAGAATTAAGTTGGTTAG 49 139  457 3 233702 6 474GGTGCTCCTGAGCCCCAGCT 82 140 n/a n/a 233703 6 488 TGATGTTACTTCTGGGTGCT 63141  494 2 233704 6 511 TGCTGTTCTACAAAACTTTT 79 142  517 3 233705 6 535AGGAGTTCTCTTATGCTGTT 86 143 n/a n/a 233706 6 540 TCTGGAGGAGTTCTCTTATG 58144 n/a n/a 233707 6 545 CACACTCTGGAGGAGTTCTC 77 145 n/a n/a 233708 6632 GGGTTCTTGAATACCAGTCT 58 146  638 2 233709 6 649 GAAAGAGAATTTTCTGAGGG29 147  655 3 233710 6 677 AGTAGTTCTTGGTGCTCTTG 68 62  683 0 233711 6730 GCAGGAAGGTCATCTTGTTC 62 148 n/a n/a 233712 6 739GAGCAGTCGGCAGGAAGGTC 62 149 n/a n/a 233713 6 778 TACACGCCACTTGTATGTTC 55150 784 1 233714 6 787 TTAATAGTGTACACGCCACT 62 151 n/a n/a 233715 6 816AGACATTAAACCCTTGGGAG 47 152 n/a n/a 233716 6 838 CTGCCTGATTGGGTATCACA 76153 n/a n/a 233717 6 856 TGAATTAATGTCCATGGACT 61 75  862 0 233718 6 871CCATCTTTCCGGTGTTGAAT 64 154  877 3 233719 6 884 GAAGTCCTGTGAGCCATCTT 69155 n/a n/a 233720 6 935 TTCTCCATCGAGCCTCCCAA 56 156  941 1 233721 6 953CTCTAGGCCCAACCAAAATT 67 82  959 0 233722 6 958 ATCTTCTCTAGGCCCAACCA 6383  964 0 233723 6 975 GTTGGACTATAGCATAGATC 70 157 n/a n/a 233724 6 988ATGTAGTTAGACTGTTGGAC 57 158 n/a n/a 233725 6 1033 ACGTAGTGCTTGCTGTCTTT81 159 n/a n/a 233726 6 1055 GCCCAGGTGAAAGGAGTATT 70 160 n/a n/a 2337276 1081 TGTAGCGTGTAGTTGGTTTC 38 161 1087 1 233728 6 1086CCACATGTAGCGTGTAGTTG 59 162 1092 3 233729 6 1091 CTCAGCCACATGTAGCGTGT 57163 n/a n/a 233730 6 1096 GCAATCTCAGCCACATGTAG 62 164 n/a n/a 233731 61138 ATCAGGTCTGTGTGCTCTGG 75 165 n/a n/a 233732 6 1149ATGTAGAAAACATCAGGTCT 66 166 n/a n/a 233733 6 1160 TCTGTGATTCCATGTAGAAA61 167 1166 3 233734 6 1186 TCTGGACAGTAGAGCTGTCC 46 168 1192 3 233735 61191 AACTTTCTGGACAGTAGAGC 58 169 n/a n/a 233736 6 1209ACCACCAGCCACCTGAGTAA 60 170 1215 2 233737 6 1229 TTCTCCACATATGTCATTCC 56171 n/a n/a 233738 6 1236 GGTTGTTTTCTCCACATATG 70 172 n/a n/a 233739 61277 TGGTCTGGATTTGGTTCTGG 71 173 n/a n/a 233740 6 1283TCTCTCTGGTCTGGATTTGG 73 174 n/a n/a 233741 6 1324 TAGAGCTTTCTGCTCTGAGG64 175 n/a n/a 233742 6 1363 GTGGTGGGCTGGAGCATCAT 57 176 n/a n/a 2337436 1376 TGAAGCTTCTTAGGTGGTGG 55 177 n/a n/a 233744 6 1390TGTCTCAGTTCAGTTGAAGC 60 178 n/a n/a 233745 6 1430 TCGGGAGGACTTTAATATTT50 179 n/a n/a 233746 7 13 GGAACTTCTCCCTCCTGTCC 49 180 n/a n/a 233747 8202 TAACAATGAGTTTAAACCTA 17 181 n/a n/a 233748 8 210TCTGATCTTAACAATGAGTT 0 182 n/a n/a

Example 3 Design and Screening of Duplexed Oligomeric CompoundsTargeting Angiopoietin-Like 3

In accordance with the invention, a series of duplexes, including dsRNAand mimetics thereof, comprising oligomeric compounds of the inventionand their complements can be designed to target angiopoietin-like 3. Thenucleobase sequence of the antisense strand of the duplex comprises atleast a portion of an oligonucleotide targeted to angiopoietin-like 3 asdisclosed herein. The ends of the strands may be modified by theaddition of one or more natural or modified nucleobases to form anoverhang. The sense strand of the nucleic acid duplex is then designedand synthesized as the complement of the antisense strand and may alsocontain modifications or additions to either terminus. The antisense andsense strands of the duplex comprise from about 17 to 25 nucleotides, orfrom about 19 to 23 nucleotides. Alternatively, the antisense and sensestrands comprise 20, 21 or 22 nucleotides.

For example, in one embodiment, both strands of the dsRNA duplex wouldbe complementary over the central nucleobases, each having overhangs atone or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (incorporated herein as SEQ ID NO: 183) and having atwo-nucleobase overhang of deoxythymidine (dT) would have the followingstructure:

  cgagaggcggacgggaccgTT Antisense Strand (SEQ ID NO: 184)  ||||||||||||||||||| TTgctctccgcctgccctggc Complement (SEQ ID NO: 185)

Overhangs can range from 2 to 6 nucleobases and these nucleobases may ormay not be complementary to the target nucleic acid. In anotherembodiment, the duplexes can have an overhang on only one terminus.

In another embodiment, a duplex comprising an antisense strand havingthe same sequence, for example CGAGAGGCGGACGGGACCG (SEQ ID NO: 183), canbe prepared with blunt ends (no single stranded overhang) as shown:

cgagaggcggacgggaccg Antisense Strand (SEQ ID NO: 183)||||||||||||||||||| gctctccgcctgccctggc Complement (SEQ ID NO: 186)

The RNA duplex can be unimolecular or bimolecular; i.e, the two strandscan be part of a single molecule or may be separate molecules.

RNA strands of the duplex can be synthesized by methods routine to theskilled artisan or purchased from Dharmacon Research Inc. (Lafayette,Colo.). Once synthesized, the complementary strands are annealed. Thesingle strands are aliquoted and diluted to a concentration of 50 μM.Once diluted, 30 μL of each strand is combined with 154, of a 5×solution of annealing buffer. The final concentration of said buffer is100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesiumacetate. The final volume is 75 μL. This solution is incubated for 1minute at 90° C. and then centrifuged for 15 seconds. The tube isallowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes areused in experimentation. The final concentration of the dsRNA duplex is20 μM.

Once prepared, the duplexed compounds are evaluated for their ability tomodulate angiopoietin-like 3. When cells reached 80% confluency, theyare treated with duplexed compounds of the invention. For cells grown in96-well plates, wells are washed once with 200 μL OPTI-MEM-1™reduced-serum medium (Gibco BRL) and then treated with 130 μL ofOPTI-MEM-1™ containing 12 μg/mL LIPOFECTIN™ (Gibco BRL) and the desiredduplex antisense compound at a final concentration of 200 nM (a ratio of6 μg/mL LIPOFECTIN™ per 100 nM duplex antisense compound). After 5 hoursof treatment, the medium is replaced with fresh medium. Cells areharvested 16 hours after treatment, at which time RNA is isolated andtarget reduction measured by RT-PCR.

Example 4 Antisense Inhibition of Mouse Angiopoietin-Like 3 byOligomeric Compounds: Dose Response Studies

In a further embodiment of the present invention, three oligonucleotideswere selected for additional dose-response studies. Mouse primaryhepatocytes were treated with 6.25, 25, 100, or 400 nM of ISIS 233693(SEQ ID NO: 131), ISIS 233698 (SEQ ID NO: 136), or ISIS 233725 (SEQ IDNO: 159), or the scrambled control oligonucleotide ISIS 113529 (5-10-5gapmer, CTCTTACTGTGCTGTGGACA, incorporated herein as SEQ ID NO: 11) andmRNA levels were measured as described in other examples herein.Untreated cells served as the control to which the data were normalized.

Results of these studies are shown in Table 6. Data are averages fromthree experiments and are expressed as percent inhibition relative tountreated control.

TABLE 6 Inhibition of angiopoietin-like 3 mRNA expression in mouseprimary hepatocytes % Inhibition Dose (nM) Treatment 6.25 25 100 400ISIS 233693 45 77 87 87 ISIS 233698 0 13 33 52 ISIS 233725 28 65 66 79ISIS 113529 0 0 0 0

As shown in Table 6, ISIS 233693, 233698, and 233725 reducedangiopoietin-like 3 mRNA levels in a dose-dependent manner.

Example 5 Effects of Antisense Inhibition of Angiopoietin-Like 3: InVivo Studies in C57BL/6 Mice

In accordance with the present invention, two oligonucleotides targetingmouse angiopoietin-like 3 were chosen for in vivo studies. Male C57BL/6mice fed normal chow were injected twice weekly with 50 mg/kg doses ofeither ISIS 233693 (SEQ ID NO: 131) or ISIS 233698 (SEQ ID NO: 136) fortwo weeks. Each treatment group was comprised of 5 animals. A group ofanimals received injections of saline twice weekly for 2 weeks. Thissaline-injected group served as the control group to which theoligonucleotide-treated groups were compared.

After the 2 week treatment period, the mice were sacrificed andangiopoietin-like 3 mRNA levels were evaluated in liver. mRNA expressionlevels were quantitated by real-time PCR as described in other examplesherein. Relative to saline-treated mice, ISIS 233693 caused a 44%decrease in angiopoietin-like 3 mRNA levels. ISIS 233698 caused a 41%decrease in angiopoietin-like 3 mRNA levels. The data demonstrate thatangiopoietin-like 3 antisense oligonucleotide treatment can effectivelyinhibit target mRNA expression in liver.

Spleen weight, body weight, and liver weight were measured at the end ofthe study. Average tissue and body weights measured (in grams) at theend of the treatment period are shown in Table 7. As shown in Table 7,body weight, liver weight and spleen weight were not affected byoligonucleotide treatment.

TABLE 7 Effect of antisense inhibition of angiopoietin-like 3 expressionon tissue and body weights in lean mice Body Liver Spleen TreatmentWeight Weight Weight Saline 24 1.1 0.1 ISIS 233693 23 1.1 0.1 ISIS233698 22 1.0 0.1

At study termination, the animals were evaluated for serum cholesterol,HDL, LDL, triglyceride and glucose levels by routine analysis using anOlympus Clinical Analyzer (Olympus America Inc., Melville, N.Y.). Theserum transaminases ALT and AST, increases in which can indicatehepatotoxicity, were also measured. Levels of AST or ALT associated withliver toxicity were not observed. The levels of serum glucose (GLUC),cholesterol (CHOL), LDL, HDL, and triglycerides (TRIG) measured arepresented in Table 8 as the average result from each treatment group inmg/dL.

TABLE 8 Effect of antisense inhibition of angiopoietin-like 3 expressionon serum glucose, lipids, and transaminases in lean mice Treatment CHOLGLU HDL TG LDL Saline 80 172 58 109 14 ISIS 233693 70 251 53 72 10 ISIS233698 87 266 67 84 12

As shown in Table 8, treatment with ISIS 233693 and ISIS 233698 reducedserum triglycerides. Treatment with ISIS 233693 reduced totalcholesterol.

Example 6 Effects of Antisense Inhibition of Angiopoietin-Like 3: InVivo Dose-Response Studies in High-Fat Fed Mice

The C57BL/6 mouse strain is reported to be susceptible tohyperlipidemia-induced atherosclerotic plaque formation. Accordingly,these mice were fed a high-fat diet and used in the following studies toevaluate the effects of angiopoietin-like 3 antisense oligonucleotideson mRNA expression.

Male C57BL/6 mice were placed on a high-fat diet containing 60% caloriesfrom fat (for example, Research Diet D12492, Research Diets Inc., NewBrunswick, N.J.). Mice receiving the high-fat diet were divided intotreatment groups. Three groups received twice-weekly injections of ISIS233693 (SEQ ID No: 131) at doses of 10 mg/kg, 25 mg/kg or 50 mg/kg, for6 weeks. Three additional groups received twice-weekly injections ofISIS 233698 (SEQ ID No: 136) at doses of 10 mg/kg, 25 mg/kg or 50 mg/kg,for 6 weeks.

A group of high-fat fed animals received injections of saline twiceweekly for 6 weeks. This saline-injected group served as the controlgroup to which the oligonucleotide-treated groups were compared.

After the 6 week treatment period, the mice were sacrificed andangiopoietin-like 3 mRNA levels were evaluated in liver. mRNA expressionlevels were quantitated by real-time PCR as described in other examplesherein. Results are presented in Table 9 as the average percentageinhibition relative to saline-treated control.

TABLE 9 Antisense inhibition of angiopoietin-like 3 expression in liverfrom high-fat fed mice: dose-response study Treatment % Inhibition ISIS233693, 10 mg/kg 73 ISIS 233693, 25 mg/kg 88 ISIS 233693, 50 mg/kg 93ISIS 233698, 10 mg/kg 17 ISIS 233698, 25 mg/kg 39 ISIS 233698, 50 mg/kg55

These data show that antisense oligonucleotides targeted toangiopoietin-like 3 mRNA effectively reduce target mRNA expression inliver in a dose-dependent manner.

Body weight was monitored throughout the study. Spleen weight, fat padweight, and liver weight were measured at the end of the study. Averagetissue and body weights measured at the end of the treatment period areshown in Table 10.

TABLE 10 Antisense inhibition of angiopoietin-like 3 expression ontissue and body weights in high-fat fed mice: dose-response study BodyTreatment Weight Liver Spleen Fat Pad Saline 36 1.2 0.1 2.1 ISIS 233693,10 mg/kg 37 1.5 0.1 2.0 ISIS 233693, 25 mg/kg 34 1.4 0.1 1.3 ISIS233693, 50 mg/kg 32 1.5 0.1 1.1 ISIS 233698, 10 mg/kg 38 1.4 0.1 1.8ISIS 233698, 25 mg/kg 33 1.2 0.1 1.5 ISIS 233698, 50 mg/kg 33 1.5 0.11.5

These data demonstrate that body weight, spleen weight and liver weightwere not affected. Fat pad weight was reduced in a dose-dependent mannerby treatment with ISIS 233698. Treatment with ISIS 233698 also reducedfat pad weight.

At study termination, the animals were evaluated for serum cholesterol,triglyceride and glucose levels by routine analysis using an OlympusClinical Analyzer (Olympus America Inc., Melville, N.Y.). The serumtransaminases ALT and AST, increases which can indicate hepatotoxicity,were also measured using an Olympus Clinical Analyzer (Olympus AmericaInc., Melville, N.Y.). The levels of serum cholesterol (CHOL) andtriglycerides (TRIG) measured are presented in Table 11 as the averageresult from each treatment group in mg/dL. Also shown are the averagelevels of HDL and LDL as well as average glucose levels (GLUC). ALT andAST, also shown in Table 11, are likewise shown as the average resultfrom each treatment group, in international units/L (IU/L).

TABLE 11 Effects of antisense inhibition of angiopoietin-like 3 on serumglucose, cholesterol, triglycerides, and liver transaminases in high-fatfed mice Treatment ALT AST CHOL HDL LDL TRIG GLUC Saline 22 47 177 14331 120 244 ISIS 233693, 23 60 151 127 23 81 263 10 mg/kg ISIS 233693, 2162 125 106 18 57 254 25 mg/kg ISIS 233693, 52 79 147 125 22 44 206 50mg/kg ISIS 233698, 28 55 151 120 27 101 285 10 mg/kg ISIS 233698, 16 53135 110 23 89 248 25 mg/kg ISIS 233698, 192 175 158 126 31 77 215 50mg/kg

As shown in Table 11, as compared to saline-treatment, treatment withISIS 233693 or ISIS 233698 resulted in decreased cholesterol levels anddose-dependent decreases in serum triglycerides. ISIS 233693 and ISIS233698 also resulted in a slight decrease in HDL which is commonlyobserved in mice when hypolipidemic agents are tested due to the factthat mice, unlike humans and other species, carry 90% of their serumcholesterol as HDL particles. Furthermore, treatment with ISIS 233693decreased LDL.

Example 7 Effects of Antisense Inhibition of Angiopoietin-Like 3 LevelsIn Vivo: Liver Triglycerides

Hepatic steatosis refers to the accumulation of lipids in the liver, or“fatty liver”, which is frequently caused by alcohol consumption,diabetes and hyperlipidemia and can progress to end-stage liver damage.Given the deleterious consequences of a fatty liver condition, it is ofuse to identify compounds that prevent or ameliorate hepatic steatosis.Hepatic steatosis may be evaluated both by measurement of tissuetriglyceride content and by histologic examination of liver tissue.

In a further embodiment, liver tissue triglyceride content was assessedin the animals described in Example 6. Liver tissue triglyceride contentwas measured using the Triglyceride GPO assay (Roche Diagnostics,Indianapolis, Ind.). Results for each treatment group were normalized tosaline-treated control and are presented in Table 12.

TABLE 12 Effects of antisense inhibition of angiopoietin-like 3 on livertriglycerides in high-fat fed mice Treatment % Control ISIS 233693, 10mg/kg 82 ISIS 233693, 25 mg/kg 54 ISIS 233693, 50 mg/kg 31 ISIS 233698,10 mg/kg 55 ISIS 233698, 25 mg/kg 41 ISIS 233698, 50 mg/kg 47

As shown in Table 12, treatment with antisense oligonucleotides targetedto angiopoietin-like 3 results in dose-dependent reduction in livertriglycerides.

Example 8 Effects of Antisense Inhibition of Angiopoietin-Like 3: InVivo Studies with ISIS 233693 in High-Fat Fed Mice

In a study similar to that described in Example 6, male C57BL/6 micewere placed on a high-fat diet containing 60% calories from fat (forexample, Research Diet D12492, Research Diets Inc., New Brunswick,N.J.). Mice receiving the high-fat diet were divided into treatmentgroups. One group received twice-weekly injections of ISIS 233693 (SEQID No: 131) at doses of 50 mg/kg, for 6 weeks.

Oligonucleotides were dissolved in saline for injection. A group ofhigh-fat fed animals received injections of saline twice weekly for 6weeks. This saline-injected group served as the control group to whichthe oligonucleotide-treated groups were compared.

After the 6 week treatment period, the mice were sacrificed andangiopoietin-like 3 mRNA levels were evaluated in liver. mRNA expressionlevels were quantitated by real-time PCR as described in other examplesherein. ISIS 233693 caused an 88% reduction in target mRNA levels.

Body weight was monitored throughout the study. Spleen weight, fat padweight, and liver weight were measured at the end of the study. Theaverage body weight, liver weight, spleen weight, and fat pad weight foranimals treated with saline alone were 33 g, 1.2 g, 0.1 g, 0.7 g,respectively. The average body weight, liver weight, spleen weight, andfat pad weight for animals treated with ISIS 233693 were 31 g, 1.6 g,0.2 g, and 0.2 g, respectively. Treatment with ISIS 233693 reduced fatpad weight by 71%.

At study termination, the animals were evaluated for serum cholesterol,triglyceride and glucose levels by routine clinical analyses (forexample at a clinical testing facility such as BTS, a division of LabCorp, San Diego, Calif.). The serum transaminases ALT and AST, and serumprotein, increases in which can indicate hepatotoxicity, and levels ofbilirubin, increases in which can indicate kidney toxicity, were alsomeasured. Toxic increases as indicators of aberrant kidney or liverfunction were not observed with ISIS 233693 treatment. ISIS 233693caused a reduction in serum triglyceride and glucose levels, but did notalter serum cholesterol, LDL, or HDL levels.

Hepatic steatosis refers to the accumulation of lipids in the liver, or“fatty liver”, which is frequently caused by alcohol consumption,diabetes and hyperlipidemia and can progress to end-stage liver damage.Given the deleterious consequences of a fatty liver condition, it is ofuse to identify compounds that prevent or ameliorate hepatic steatosis.Hepatic steatosis may be evaluated both by measurement of tissuetriglyceride content and by histologic examination of liver tissue.

Liver tissue triglyceride content was measured using the TriglycerideGPO assay (Roche Diagnostics, Indianapolis, Ind.). Average results forthe ISIS 233693 treatment group were normalized to saline-treatedcontrol. Treatment with ISIS 233693 caused a 75% reduction in livertriglyceride levels.

Histological analysis was conducted by routine procedures. Briefly,liver samples were procured, fixed in 10% neutral buffered formalin andprocessed for H&E staining and evaluation of liver morphology.Alternatively, liver tissue was procured, frozen, sectioned, andsubsequently stained with oil red 0 stain to visualize lipid depositsand counterstained with eosin to mark cytoplasm. The prepared sampleswere evaluated by light microscopy.

As assessed by oil-red 0 stain and histological analysis, livers fromanimals treated with ISIS 233693 presented with reduced fat content ascompared to saline-treated control livers.

Therefore, oligomeric compounds targeted to angiopoietin-like 3ameliorate hepatic steatosis as evaluated both by measurement of tissuetriglyceride content and by histologic examination of liver tissue.

Taken together, the in vivo studies shown herein indicate that antisenseoligonucleotide reduction of angiopoietin-like 3 results in dosedependent reductions in liver target mRNA, as well as reductions inserum and liver triglyceride levels. In addition, antisenseoligonucleotides targeted to angiopoietin-like 3 caused decreases inserum cholesterol levels in both lean and high-fat fed mice.Furthermore, reduction in fat pad weight was observed without similarreductions in body or organ weight, indicating target-specific reductionin fat content. Therefore, another aspect of the invention is a methodof reducing serum cholesterol, serum triglycerides, liver triglyceridesor fat pad weight for conditions such as hyperlipidemia.

Example 9 Effects of Antisense Inhibition of Angiopoietin-Like 3 onAtherosclerosis: Treatment with ISIS 233693 in LDLr^(−/−) Mice

The effect of ISIS 233693 as an anti-atherosclerotic agent was evaluatedin LDL receptor knockout mice fed on a hypercholesterolemic diet; amodel used for studying atherosclerosis (Ishibashi et al, J Clin.Invest. 1994 May; 93:1885-93).

Treatment

C57B1/6 mice with LDL receptor gene knockout (Jackson Labs, #2207) werefed a Harlan Tekland diet, TD 94059 (37% kCal fat, half from cocoabutter, 1.25% cholesterol). Four weeks after the initiation of the diet,the mice were divided into two groups consisting of 6-8 mice each fortreatment. The first group received twice-weekly subcutaneous injectionsof ISIS 233693 (SEQ ID No: 131) at doses of 25 mg/kg, for 16 weeks. Thesecond group received twice-weekly subcutaneous injections of mismatchedcontrol oligonucleotide, ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, incorporatedherein as SEQ ID NO: 187) at doses of 25 mg/kg, for 16 weeks.

Oligonucleotides were dissolved in saline for injection. A group ofhigh-fat fed animals received injections of saline twice weekly for 16weeks. This saline-injected group served as the control group to whichthe oligonucleotide-treated groups were compared.

Blood samples were taken every 4 weeks. At the end of the treatmentperiod, the mice were euthanized and liver and aorta were collected forfurther analysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis ofANGPTL3 using an ANGPTL3 primer probe set (forward sequenceCACCTGGGCAGTCACGAAA, designated herein as SEQ ID NO: 188; reversesequence GGAGGGCCCCAGGGATAT, designated herein as SEQ ID NO: 189; probesequence CACGCTACATGTGGCTGAGATTCGTGG, designated herein as SEQ ID NO:190). Results are presented as percent inhibition of murine ANGPTL3,relative to PBS control. As shown in Table 13, treatment with ISIS233693 resulted in significant reduction of ANGPTL3 mRNA in comparisonto the PBS control. Treatment with the control oligonucleotide, ISIS141923 did not result in significant reduction of ANGPTL3, as expected.

TABLE 13 Inhibition of ANGPTL3 mRNA in LDLr^(−/−) mouse liver relativeto the PBS control % inhibition ISIS 141923 13 ISIS 233693 90

Cholesterol and Lipid Levels

Plasma and liver triglycerides, and cholesterol were extracted by themethod of Bligh and Dyer (Bligh, E and Dyer, W, Can J Biochem Physiol,37, 911-917, 1959) and measured with the use of a commercially availabletriglyceride kit (DCL Triglyceride Reagent; Diagnostic Chemicals Ltd.).The results are presented in Tables 14-17. Table 14 demonstrates thattreatment with ISIS 233693 resulted in significant decrease incholesterol levels by 58% on week 16 compared to the PBS control. Thedecrease in total cholesterol levels was the result of significantdecrease in LDL cholesterol levels by 58% compared to the control, aspresented in Table 15. Table 16 shows a decrease in HDL that is likelymouse model dependent, as detailed previously. Similarly, Table 17demonstrates that treatment with ISIS 233693 decreased triglyceridelevels by 75% on week 16 compared to the PBS control.

TABLE 14 Effect on total cholesterol levels (mg/dL) in LDLr^(−/−) miceWeek 0 Week 4 Week 8 Week 12 Week 16 PBS 1,313 1,618 1,398 1,083 1,629ISIS 141923 1262 1710 1624 1102 1167 ISIS 233693 1353 1070 930 558 683

TABLE 15 Effect on LDL cholesterol levels (mg/dL) in LDLr^(−/−) miceWeek 0 Week 4 Week 8 Week 12 Week 16 PBS 1,031 1,120 1,001 909 1,204ISIS 141923 1022 1185 1116 902 843 ISIS 233693 1075 731 648 453 511

TABLE 16 Effect on HDL cholesterol levels (mg/dL) in LDLr^(−/−) miceWeek 0 Week 4 Week 8 Week 12 Week 16 PBS 154 166 169 145 300 ISIS 141923139 171 179 171 278 ISIS 233693 153 116 103 97 159

TABLE 17 Effect on triglyceride levels (mg/dL) in LDLr^(−/−) mice Week 0Week 4 Week 8 Week 12 Week 16 PBS 230 177 134 140 268 ISIS 141923 180154 164 156 159 ISIS 233693 191 68 61 72 67

Atherosclerotic Lesion Assessment

Atherosclerotic lesion severity was assessed in the aortae harvestedfrom mice after perfusion with PBS, followed by formalin PBS solution(5% formalin in PBS). The entire mouse aorta was dissected from theproximal ascending aorta to the bifurcation of the iliac artery by usinga dissecting microscope. Adventitial fat was removed, and the aorta wasopened longitudinally, pinned flat onto black dissecting wax, stainedwith Sudan IV, and photographed at a fixed magnification. Thephotographs were digitized, and total aortic areas and lesion areas werecalculated by using Adobe Photoshop version 7.0 and NIH Scion Imagesoftware (http://rsb.info.nih.gov/nih-image/Default.html). The results,presented in Table 18, are reported as a percentage of the total aorticarea that contained lesions. As presented, treatment with ISIS 233693resulted in a significant decrease in aortic lesions and improvement ofthe atherosclerotic condition.

TABLE 18 Effect on lesion area (% of total aortic area) in LDLr^(−/−)mice lesion area (%) PBS 44 ISIS 141923 40 ISIS 233693 18

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Tables 19and 20 expressed in IU/L. The measurements were conducted every fourweeks. Week 0 is at the start of treatment and four weeks after theinitiation of the high fat diet.

TABLE 19 Effect on liver ALT (IU/L) of LDLr^(−/−) mice Week 0 Week 4Week 8 Week 12 Week 16 PBS 29 35 39 36 36 ISIS 141923 30 32 32 33 60ISIS 233693 20 48 70 83 91

TABLE 20 Effect on liver AST (IU/L) of LDLr^(−/−) mice Week 0 Week 4Week 8 Week 12 Week 16 PBS 49 63 61 82 73 ISIS 141923 57 57 61 60 81ISIS 233693 51 60 90 95 108

Example 10 Effect of Antisense Inhibition of Angiopoietin-Like 3 onHuman apoB100 Transgenic LDLr^(−/−) Mice

The effect of ISIS 233693 as a lipid lowering agent was evaluated inhuman apoB-100 transgenic LDL receptor knockout mice fed on ahypercholesterolemic diet. The mice used in these studies have beendescribed in a previous publication Sanan et al, Proc. Natl. Acad. Sci.USA. 95: 4544-4549). In brief, this mouse strain is a hybrid crossbetween the LDLr^(−/−) mouse described originally by Ishibashi et al.(J. Clin. Invest. 92: 883-893), which is a hybrid of the 129sv andC57BL/6 strains, and the human apoB-100 transgenic mouse (Linton et al,J. Clin. Invest. 92: 3029-3037), which is a hybrid of the SJL andC57BL/6B strains. Breeding indicated that the LDLr^(−/−) and apoBoverexpression traits were homozygous, and the mice exhibited a massiveincrease in apoB-100-containing LDL.

Treatment

The mice were fed a Harlan Tekland diet, TD 88137 or ‘Western diet’ (21%anhydrous milkfat (butterfat), 34% sucrose, and a total of 0.2%cholesterol). One week after the initiation of the diet, the mice weredivided into groups consisting of 5 mice each for treatment. The firstcohort received twice-weekly subcutaneous injections of ISIS 233693 (SEQID No: 131) at doses of 12.5 mg/kg, 25 mg/kg or 50 mg/kg for 4 weeks.The second cohort received twice-weekly subcutaneous injections ofcontrol oligonucleotide, ISIS 141923 (SEQ ID NO: 187) at doses of 12.5mg/kg or 50 mg/kg, for 4 weeks.

Oligonucleotides were dissolved in saline for injection. A group ofhigh-fat fed animals received injections of saline twice weekly for 4weeks. This saline-injected group served as the control group to whichthe oligonucleotide-treated groups were compared.

The mice were weighed weekly. At the end of the treatment period, themice were euthanized and blood samples, liver, kidney, spleen and fatpads were collected for further analysis.

RNA analysis

RNA was extracted from liver tissue for real-time PCR analysis ofANGPTL3 using an ANGPTL3 primer probe set (forward sequenceCACCTGGGCAGTCACGAAA, designated herein as SEQ ID NO: 187; reversesequence GGAGGGCCCCAGGGATAT, designated herein as SEQ ID NO: 188; probesequence CACGCTACATGTGGCTGAGATTCGTGG, designated herein as SEQ ID NO:189) and of ApoCIII mRNA using an ApoCIII primer probe set (Forward:TGCAGGGCTACATGGAACAA, incorporated herein as SEQ ID NO: 12; Reverse:CGGACTCCTGCACGCTACTT, incorporated herein as SEQ ID NO: 13; Probe:CTCCAAGACGGTCCAGGATGCGC, incorporated herein as SEQ ID NO: 14). Resultsare presented as percent inhibition of murine ANGPTL3 and murineApoCIII, relative to PBS control. As shown in Table 21, treatment withISIS 233693 and ISIS 233725 resulted in significant dose-dependentreduction of ANGPTL3 mRNA in comparison to the PBS control. Treatmentwith ISIS 233693 also resulted in inhibition of ApoCIII mRNA at 50mg/kg/week.

RNA levels of liver fatty acid binding protein (LFABP) were alsomeasured by real-time PCR. The results are presented in Table 21, anddemonstrate that inhibition of ANGPTL3 by ISIS oligonucleotides alsoinfluences the transport of fatty acids in the liver, by inhibitingLFABP. Treatment with the control oligonucleotide, ISIS 141923 did notresult in significant reduction of ANGPTL3, LFABP or apoCIII, asexpected.

TABLE 21 Percent inhibition of ANGPTL3 and LFABP mRNA in mouse liverrelative to the PBS control Dose ISIS No (mg/kg) ANGPTL3 ApoCIII LFABP233693 50 95 39 53 25 90 25 12 12.5 77 8 10 233725 50 91 27 80 25 79 2751 12.5 62 12 11

Cholesterol and Lipid Levels

Plasma and liver triglycerides, and cholesterol were extracted by themethod of Bligh and Dyer (Bligh, E and Dyer, W, Can J Biochem Physiol,37, 911-917, 1959) and measured with the use of a commercially availabletriglyceride kit (DCL Triglyceride Reagent; Diagnostic Chemicals Ltd.).The results are presented in Table 22. The study demonstrates thattreatment with ISIS 233693 and ISIS 233725 decreased cholesterol levelsby 63% and 37% respectively, at 25 mg/kg/week compared to the PBScontrol. The decrease in total cholesterol levels was mainly the resultof significant decreases in LDL cholesterol levels by 63% and 34%respectively, compared to the control, as presented. The slight decreasein HDL may be mouse model dependent as detailed previously as HDLlowering is commonly observed in mice when hypolipidemic agents aretested in mice. The study demonstrates that treatment with ISIS 233693and ISIS 233725 decreased triglyceride levels by 82% and 70%respectively, at 25 mg/kg/week compared to the PBS control.

Therefore, treatment with ISIS oligonucleotides targeting ANGPTL3 causessignificant improvements of plasma lipid profile in this mouse model.

TABLE 22 Effect on plasma lipid levels (mg/dL) Dose Total (mg/kg)cholsterol HDL LDL Triglycerides PBS 2534 430 1591 1022 ISIS 141923 501730 347 998 945 12.5 2489 412 1512 1168 ISIS 233693 50 982 256 585 24325 944 245 596 184 12.5 1480 307 990 281 ISIS 233725 50 1407 325 862 40325 1587 312 1052 309 12.5 2060 370 1366 522

Glucose

To evaluate the effect of ISIS oligonucleotides on glucose production,plasma glucose values were determined using a Beckman Glucose AnalyzerII (Beckman Coulter) by a glucose oxidase method. The results arepresented in Table 23 and demonstrate that treatment with ISIS 233693and ISIS 233725 resulted in decrease of plasma glucose levels both by40% at 50 mg/kg/week compared to the PBS control.

TABLE 23 Effect on plasma glucose levels (mg/dL) Dose (mg/kg) GlucosePBS 341 ISIS 141923 50 334 12.5 315 ISIS 233693 50 203 25 261 12.5 255ISIS 233725 50 203 25 280 12.5 284

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Table 24expressed in IU/L.

As demonstrated by this study, treatment with ISIS 233693 did not causeany significant increase in transaminase levels and therefore, did notresult in any adverse effect on liver function. This assay as well asthe RNA analysis establishes ISIS 233693 to be a potent and tolerableantisense oligonucleotide targeting ANGPTL3.

TABLE 24 Effect on plasma transaminases (IU/L) Dose (mg/kg) ALT AST PBS57 80 ISIS 141923 50 61 88 12.5 43 79 ISIS 233693 50 138 152 25 43 7012.5 45 87 ISIS 233725 50 1443 736 25 217 194 12.5 88 138

Body and Organ Weights

To evaluate the effect of ISIS oligonucleotides on organ weight, organswere harvested and weight taken after termination of the study. Theresults are presented in Table 25 and demonstrate that treatment withISIS oligonucleotides has no effect on liver, spleen or kidney weights.Treatment with ISIS 233693 did decrease fat pad weights of the mice by77% at 50 mg/kg/week compared to the PBS control.

TABLE 25 Effect on organ weights (g) Dose mg/kg/wk) Liver Kidney SpleenFat PBS 1.45 0.35 0.09 1.49 ISIS 141923 50 1.42 0.33 0.11 0.70 12.5 1.390.32 0.09 0.95 ISIS 233693 50 1.65 0.33 0.15 0.35 25 1.42 0.32 0.13 0.5112.5 1.49 0.35 0.11 0.89

Example 11 Effects of Antisense Inhibition of ANGPTL3 Compared toFenofibrate Inhibition in C57BL/6 Mice

ISIS 233693 targeting mouse ANGPTL3 in comparison to fenofibrate wasevaluated in naïve C57BL/6 mice. Fenofibrate is a commercially availabletreatment for hypercholesterolemia and hypertriglyceridemia in subjectsand is known to reduce cholesterol, low-density lipoprotein (LDL), verylow density lipoprotein (VLDL) and triglycerides levels, as well asincrease high-density lipoprotein (HDL) levels.

A group of five male C57BL/6 mice fed normal chow were injected twiceweekly with 50 mg/kg doses of ISIS 233693 (SEQ ID NO: 131) for sixweeks. A second group of mice was treated with fenofibrate, administeredas a daily gavage of 50 mg/kg/week. A group of animals receivedinjections of PBS twice weekly for 6 weeks. This PBS-injected groupserved as the control group to which the oligonucleotide-treated groupswere compared.

After the 6 week treatment period, the mice were sacrificed and ANGPTL3mRNA levels were evaluated in liver. The mRNA expression levels werequantitated by real-time PCR, as described in other examples herein.Relative to PBS-treated mice, ISIS 233693 caused an 85% decrease inANGPTL3 mRNA levels. The data demonstrate that ANGPTL3 antisenseoligonucleotide treatment can effectively inhibit target mRNA expressionin liver.

Cholesterol and Lipid Levels

Plasma and liver triglycerides and cholesterol were extracted by themethod of Bligh and Dyer (Bligh, E and Dyer, W, Can J Biochem Physiol,37, 911-917, 1959) and measured with the use of a commercially availabletriglyceride kit (DCL Triglyceride Reagent; Diagnostic Chemicals Ltd.).The results are presented in Table 26. The study demonstrates thattreatment with ISIS 233693 decreased cholesterol levels by 23% at 50mg/kg/week compared to the PBS control. The study demonstrates thattreatment with ISIS 233693 decreased triglyceride levels by 38% at 50mg/kg/week compared to the PBS control. Treatment with fenofibrate hadno effect on cholesterol or triglyceride levels.

Therefore, treatment with ISIS oligonucleotides targeting ANGPTL3 causessignificant improvements of plasma lipid profile in this mouse model.

TABLE 26 Effect on plasma lipid levels (mg/dL) Total cholesterol HDL LDLTriglycerides PBS 87 75 17 90 ISIS 233693 67 54 16 56 Fenofibrate 96 8516 94

Glucose

To evaluate the effect of ISIS oligonucleotides on glucose production,plasma glucose values were determined using a Beckman Glucose AnalyzerII (Beckman Coulter) by a glucose oxidase method. The results arepresented in Table 27 and demonstrate that treatment with ISIS 233693resulted in decrease of plasma glucose levels by 19% at 50 mg/kg/weekcompared to the PBS control. Treatment with fenofibrate had no effect onglucose levels.

TABLE 27 Effect on plasma glucose levels (mg/dL) Glucose PBS 242 ISIS233693 196 Fenofibrate 228

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Table 28expressed in IU/L.

As demonstrated by this study, treatment with either ISIS 2336393 orfenofibrate did not cause any significant increase in transaminaselevels and therefore, did not result in any adverse effect on liverfunction.

TABLE 28 Effect on plasma transaminases (IU/L) ALT AST PBS 23 83 ISIS233693 38 70 Fenofibrate 34 70

Effect on Plasma NEFA and 3HB Levels

NEFA and 3-HB levels were assayed in the mice groups and are shown inTable 29. NEFA and 3-HB levels, as indicators of fat oxidation, were notsignificantly affected by treatment with ISIS oligonucleotides.Treatment with fenofibrate increased fat oxidation, as indicated byincreases in the levels of both NEFA and 3HB.

TABLE 29 Effect on NEFA and 3HB levels NEFA 3HB PBS 0.90 393 ISIS 2336930.98 447 Fenofibrate 1.20 641

Organ Weights

To evaluate the effect of ISIS oligonucleotides on organ weight, organswere harvested and weight taken after termination of the study. Theresults are presented in Table 30 and demonstrate that treatment withISIS 233693 has no effect on liver, spleen or kidney weights. Treatmentwith ISIS 233693 did decrease white adipose tissue weight of the mice by45% at 50 mg/kg/week compared to the PBS control.

TABLE 30 Effect on organ weights (g) White adipose Liver Spleen Kidneytissue PBS 1.1 0.12 0.35 0.53 ISIS 233693 1.3 0.1 0.35 0.29 Fenofibrate1.3 0.08 0.37 0.39

Example 12 Effects of Antisense Inhibition of ANGPTL3 in Sprague-DawleyRats

ISIS 360363 (GTGACATATTCTTCACCTCT; SEQ ID NO: 191) and ISIS 360382(TTTAAGTGACGTTACCTCTG; SEQ ID NO: 192), both 5-10-5 MOE gapmerstargeting rat mRNA sequence SEQ ID NO: 193 (Genbank Accession No.XM_(—)233218.1) at start positions 333 and 476, respectively, wereevaluated in Sprague Dawley rats.

Two groups of Sprague Dawley rats fed normal chow were injected weeklywith 50 mg/kg doses of ISIS 360363 or ISIS 360382 for six weeks. A groupof animals received injections of PBS twice weekly for 6 weeks. ThisPBS-injected group served as the control group to which theoligonucleotide-treated groups were compared.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis ofANGPTL3. Results are presented as percent inhibition of rat ANGPTL3,relative to PBS control. As shown in Table 31, treatment with ISIS360363 and ISIS 360382 resulted in significant reduction of ANGPTL3 mRNAin comparison to the PBS control.

TABLE 31 Inhibition of ANGPTL3 mRNA in Sprague Dawley rat liver relativeto the PBS control % ISIS No inhibition 360363 70 360382 89

Cholesterol and Lipid Levels

Plasma triglycerides and cholesterol were extracted by the method ofBligh and Dyer (Bligh, E and Dyer, W, Can J Biochem Physiol, 37,911-917, 1959) and measured with the use of a commercially availabletriglyceride kit (DCL Triglyceride Reagent; Diagnostic Chemicals Ltd.).The results are presented in Table 26. The study demonstrates thattreatment with ISIS 360363 and 360382 decreased triglyceride levels by67% and 81% respectively, compared to the PBS control. Therefore,treatment with ISIS oligonucleotides targeting ANGPTL3 causessignificant improvements of plasma triglyceride profile in this ratmodel. Treatment with ISIS oligonucleotides in this model had no effecton total cholesterol or LDL levels.

TABLE 32 Effect on plasma triglyceride levels (mg/dL) PBS 136 ISIS360363 45 ISIS 360382 26

1-13. (canceled)
 14. An assay for identifying a compound that inhibitsangiopoietin-like 3 (ANGPTL3) comprising: a) contacting cells with acompound comprising a modified oligonucleotide 10 to 30 linkednucleosides in length targeted to ANGPTL3; b) measuring an amount ofANGPTL3 produced by the cells contacted by the compound targetingANGPTL3; and c) comparing the amount of ANGPTL3 produced from the cellscontacted with the compound targeting ANGPTL3 to an amount of ANGPTL3produced by cells not contacted with the compound targeting ANGPTL3;wherein a reduction in ANGPTL3 production by the cells contacted by thecompound targeting ANGPTL3 compared to the production of ANGPTL3produced by cells not contacted by the compound targeting ANGPTL3identifies the compound that inhibits ANGPTL3.
 15. A kit for decreasingangiopoietin-like 3 (ANGPTL3) wherein the kit comprises: a) a compoundcomprising a modified oligonucleotide 10 to 30 linked nucleosides inlength targeted to ANGPTL3, wherein the compound reduces expression ofANGPTL3 by at least 40%, and b) a label.
 16. The kit of claim 14,wherein the kit further comprises a second agent.
 17. A method fortreating cardiovascular disease in a subject, comprising: administeringa therapeutically effective amount of a compound comprising a modifiedoligonucleotide 10 to 30 linked nucleosides in length targeted toangiopoietin-like protein 3 (ANGPTL3) to a subject in need thereof. 18.The method of claim 17, comprising identifying a subject in need oftreatment of cardiovascular disease and administering the compound tothe subject.
 19. The method of claim 17, wherein expression of ANGPTL3is reduced by at least 40% in the subject.
 20. The method of claim 17,wherein the subject is a human subject.
 21. The method of claim 17,wherein the modified oligonucleotide is targeted to the 3′ UTR, 5′ UTR,exons, introns, exon/intron junctions, coding region, translationinitiation region or translation termination region of SEQ ID NO:4. 22.The method of claim 17, wherein the modified oligonucleotide has anucleobase sequence at least 90%, at least 95% or 100% complementary toSEQ ID NO: 4 as measured over the entirety of said modifiedoligonucleotide.
 23. The method of claim 17, wherein at least oneinternucleoside linkage of the modified oligonucleotide is a modifiedinternucleoside linkage.
 24. The method of claim 23, wherein the atleast one internucleoside linkage is a phosphorothioate internucleosidelinkage.
 25. The method of claim 17, wherein at least one nucleoside ofthe modified oligonucleotide comprises a modified sugar.
 26. The methodof claim 25, wherein at least one modified sugar is a bicyclic sugar.27. The method of claim 25, wherein at least one modified sugarcomprises a 2′-O-methoxyethyl, a (4′-CH(CH₃)—O-2′) BNA, a (4′-CH₂—O-2′)BNA, or a 4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or
 2. 28. The methodof claim 17, wherein at least one nucleoside of the modifiedoligonucleotide comprises a modified nucleobase.
 29. The method of claim28, wherein the modified nucleobase is a 5-methylcytosine.
 30. Themethod of claim 17, wherein the modified oligonucleotide consists of 20linked nucleosides.
 31. The method of claim 30, wherein the modifiedoligonucleotide comprises: a) a gap segment consisting of linkeddeoxynucleosides; b) a 5′ wing segment consisting of linked nucleosides;c) a 3′ wing segment consisting of linked nucleosides; wherein the gapsegment is positioned between the 5′ wing segment and the 3′ wingsegment and wherein each nucleoside of each wing segment comprises amodified sugar.
 32. The method of claim 17, wherein the compound reducesANGPTL3 expression in the subject and: a) reduces apoC-III expressionlevels; b) reduces triglyceride levels; c) reduces cholesterol levels;d) reduces LDL levels; e) reduces glucose levels; f) improves insulinsensitivity; and/or g) ameliorates a metabolic or cardiovasculardisease.
 33. The method of claim 17, wherein the compound is in a saltform.
 34. The method of claim 17, wherein the compound issingle-stranded.
 35. The method of claim 17, wherein the compoundcomprises a conjugate.
 36. The method of claim 17, wherein the compoundis in a pharmaceutical composition comprising a pharmaceuticallyacceptable diluent or carrier.
 37. The method of claim 36, wherein thecompound is in a salt form.
 38. The method of claim 17, wherein thecardiovascular disease is atherosclerosis or hepatic steatosis.
 39. Themethod of claim 17, wherein the cardiovascular disease is hyperlipidemiaand/or dyslipidemia.
 40. The method of claim 17, wherein theadministering is by parenteral administration.
 41. The method of claim40, wherein the administering is by injection.